Methods for the detection of jc polyoma virus

ABSTRACT

Methods and compositions for determining whether a subject is at risk for PML, including subjects being treated with immunosuppressants, by determining whether the subject harbors a JCV variant with reduced binding for sialic acid relative to a normal JCV, are presented. Furthermore, combinations of JCV-VP1 sequence variations that are associated with PML and that can be used as a basis of an assay for identifying subjects susceptible to PML, subjects with PML (e.g., early stage PML), or subjects at risk of developing PML in response to an immunosuppressive treatment are provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) from U.S.provisional application Ser. No. 61/150,310 entitled “Methods for theDetection of JC Polyoma Virus” filed Feb. 5, 2009, the disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods for the detection of the human JCpolyomavirus virus, diagnosis of progressive multifocalleukoencephalopathy (PML), and the development of therapeutics for PML.

BACKGROUND OF THE INVENTION

JC polyomavirus (JCV) infection in humans can cause a demyelinatingdisease of the central nervous system, progressive multifocalleukoencephalopathy (PML). However, JCV infection usually does notresult in PML in healthy subjects. JCV infection is prevalent in manyhuman populations without causing widespread PML. PML typically onlydevelops in JCV-infected subjects that also have a weakened immunesystem. Subjects that are immuno-compromised due to a disease or animmunosuppressive treatment may be vulnerable to PML associated with aJCV infection.

SUMMARY OF THE INVENTION

Aspects of the invention relate to methods and compositions fordetermining whether a subject is susceptible to PML. In particular, theinvention provides methods and compositions for determining whether asubject is at risk of developing PML if the subject's immune system iscompromised or suppressed. For example, aspects of the invention relateto determining whether a subject is suitable for an initial or continuedtreatment with an immunosuppressive agent by determining the subject'srisk profile for developing PML caused by a JCV infection.

The human genome is exposed to and may acquire many viruses during thelifetime of an individual. One example of such a virus is JCpolyomavirus (JCV). JC virus infection is highly prevalent in humans.Primary infection with JCV occurs asymptomatically during childhood(Padgett & Walker, 1973). JCV is then disseminated throughout the body,probably through viraemia (Ikegaya et al., 2004). It is thought that JCVpersist mostly in brain and renal tissue. While infection by JCV isasymptomatic in most subjects, infection may result in seriousconditions (like PML) and even death in some subjects. Subjects mostsusceptible to PML are subjects that are immuno-compromised (e.g., AIDSpatients) or subjects undergoing treatment with immuno-suppressants (forinstance after organ transplant or to treat an inflammation relatedcondition such as multiple sclerosis). The present invention providesJCV variants, identifies a panel of variants including novel variantsthat are associated with increased PML risk, and methods related todiscoveries of structural and functional connections between JCVvariants and PML.

A “wild type” JCV sequence is used herein to refer to the sequence ofany of the archetypes of JCV found in healthy subjects not having PML,and/or not being at risk for PML. In some embodiments, a consensus “wildtype” reference sequence may be an average of sequences found in a groupof healthy individuals. The discrepancy between high viral prevalenceand low incidence of PML suggests that, in addition to immunedysfunction, there could be some unique viral characteristics thatregulate the progression from the asymptomatic infection to the PML. Insome embodiments, aspects of the invention relate to the discovery thatthe part of the viral surface protein that is responsible for viralinteraction with cellular receptors and host cell infection acquiresspecific amino acid mutations in the patient somewhere en route from thekidney, the site of asymptomatic infection, to the CNS, the site of PML.Furthermore, in some embodiments PML-specific mutations change theability of the viral capsid to bind various sialic acids and a varietyof peripheral cell types but retain the ability to bind to CNS glialcells.

Based on the mathematical analysis of the published VP1 sequences fromPML and non-PML patients, a positive selection of specific amino acidvariants during PML is provided. However, since publicly available PMLsequences were obtained from CSF or brain tissues of PML patientswhereas non-PML sequences were obtained from the urine of healthysubjects, just based on the analysis of Genebank samples it could notunequivocally be demonstrated whether these amino acid variants wereproduced via a viral mutation in the patients or represented one or morerare viral variants that occur in all JCV clades and are enriched (e.g.,positively selected) in PML cases as being more likely to be causingPML. According to aspects of the invention, VP1 substitutions occurwithin the patient and can lead to PML. This is supported by theanalysis of matched urine-CSF and urine-plasma samples from the samepatient taken at the same time point. CSF and plasma VP1 sequences fromPML patients contained single amino acid substitutions or deletions ofseveral amino acids relative to VP1 sequences isolated from the urine ofthe same patient. As shown herein, JC viral types found in the CSF,plasma, and urine of the same individual were of the same viral strain,whereas different patients carried different strains. Therefore, butwithout wishing to be bound by theory, the presence of VP1 amino acidsubstitutions in the CSF, but not in urine, results from the appearanceof a mutation within a patient rather than by a dual infection with twodifferent viral variants.

Some of the amino acid substitutions detected in the CSF and plasma ofPML patients were previously described. However, these substitutions hadnot been directly correlated with increased PML risk and theirstructural and functional properties had not been associated withaspects of PML development and progression. Additional JCV VP1 mutationsand/or deletions associated with PML also are provided herein.

According to aspects of the invention, substitutions in the VP1 proteinmay be more important for determining early disease progression (e.g.,by enabling the virus to migrate from the periphery to the CNS andinfecting CNS cells) rather than determining different outcomes indifferent clinical contexts. However, in some embodiments there may bean association between the type of mutation present in VP1 and certainclinical measurements. For example, lower JCV CSF replication levelswere observed in patients with non-mutated virus or virus carryingmutations/deletions at positions 122-134. It also is possible that sincemutations affect the strength of viral interaction with cellularreceptors, viral release from dead cells also may be hindered for avirus that binds to those receptors tightly. Accordingly, a virus thathas a weaker cell binding avidity may be found to be released into theextracellular space more abundantly. This is consistent with theobservation that mutants at positions 55-61 and 265-271 lose theirability to bind to sialic acid receptors relative to non-mutated virus,and thus might have a better ability to detach from cell debris (afterthe virus kills the host cell) and find their way to the CSF.

Accordingly, even though a subject may be concurrently infected withseveral different versions of JCV (e.g., with different variants indifferent tissues or organs), PML associated mutations is more likely toarise from an existing JCV virus population in an individual that isinfected with a wild-type JCV.

Aspects of the invention are based, at least in part, on the discoverythat certain types of mutations in the JCV capsid protein are associatedwith the conversion of an asymptomatic form of JCV infection into aPML-associated form of JCV. According to aspects of the invention,without committing to any particular mechanism, mutations that disruptthe ability of a JCV particle to bind to or interact with sialic acidcan result in the virus no longer being contained in the peripheralcompartment of a subject and result in the virus obtaining freer passageto the CNS, a site of PML disease. In some embodiments, these mutationsallow virus to achieve that by avoiding “trapping” on certainglycoproteins and glycolipid pseudoreceptors expressed in peripheralorgans and cells, including but not limited to immune cells and redblood cells. Additionally, as some of these sites are targets on normalhost immune response (antibody and T-cell) these mutations may allowvirus evading recognition by the immune system, particularly in subjectswith weakened immune systems.

Aspects of the invention provide methods and compositions for evaluatinga subject's risk profile for PML.

In some embodiments, aspects of the invention relate to determiningwhether a subject has been exposed to an infection by any JCV variant.In some aspects, infection by a wild-type JCV variant may increase therisk profile for PML since PML associated mutations may arise from thewild-type JCV, even though these may be rare events. Accordingly, insome embodiments, a subject may be tested for the presence of one ormore indicia of JCV infection (e.g., detection of viral proteins ornucleic acid, or indirectly via the detection of an immune response to aJCV infection, e.g., in the form of serum antibodies to JCV viralproteins). It should be appreciated that indicia of any JCV infection(e.g., wild-type or variant) may be evaluated. If a subject has alreadybeen exposed to a JCV infection, then the subject may be identified ashaving a higher risk profile than a non-infected subject. Accordingly, aJCV-infected subject that is being treated with an immunosuppressiveagent, may be evaluated for specific JCV mutations or may be monitoredmore frequently than a non-infected subject.

In some embodiments, a subject that is being treated with (or is goingto start a treatment with) an immunosuppressive agent is tested for oneor more indicia of JCV infection. If one or more indicia of JCVinfection are detected, the subject may be evaluated for the presence ofone or more JCV variants associated with PML as described herein. If noindicia for JCV are detected, the subject may be monitored over time,e.g., every 4 weeks, monthly, every three months, every 4 months, every6 months, or every 12 months, for the presence of any indicia of JCVinfection. If a JCV infection is detected, the subject may be furtherevaluated for the presence of one or more JCV variants. If a JCV variantassociated with increased PML risk is detected, the subject may befurther monitored to detect any early signs of PML and/or the treatmentregimen may be altered as described in more detail herein.

In one aspect the invention provides a method comprising interrogating abiological sample from a subject for at least one indicium of a variantJCV VP1 capsid protein that comprises a substitution of amino acidresidue 122, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises the at least oneindicium.

In one aspect the invention provides a method comprising interrogating abiological sample from a subject for at least one indicium of a variantXV VP1 capsid protein that comprises a substitution of amino acidresidue 2, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises the at least oneindicium.

In one aspect the invention provides a method comprising interrogating abiological sample from a subject for at least one indicium of a variantJCV VP1 capsid protein that comprises a substitution of amino acidresidue 66, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises the at least oneindicium.

In one aspect the invention provides a method comprising interrogating abiological sample from a subject for at least one indicium of a variantJCV VP1 capsid protein that comprises a substitution of amino acidresidue 283, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises the at least oneindicium.

In some embodiments the method further comprises interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein that comprises a substitution of at least one of amino acidresidues 2 and 66. In some embodiments the method further comprisesinterrogating the biological sample for at least one indicium of avariant JCV VP1 capsid protein that comprises a substitution of at leastone of amino acid residues 122 and 66. In some embodiments the methodfurther comprises interrogating the biological sample for at least oneindicium of a variant JCV VP1 capsid protein that comprises asubstitution of at least one of amino acid residues 2 and 122. In someembodiments the sample is interrogated using an assay capable ofdetecting at least one indicium of each of variant JCV VP1 capsidproteins having a substitution of at least one of amino acid residues 2,66, and 122, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises at least one indicium ofat least one of the variant JCV VP1 capsid proteins. In some embodimentsthe sample is interrogated using an assay capable of detecting at leastone indicium of each of variant JCV VP1 capsid proteins having asubstitution of at least one of amino acid residues 2, 66, 122 and 283and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises at least one indicium ofat least one of the variant JCV VP1 capsid proteins.

In some embodiments the method further comprises interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein that comprises a deletion of amino acid fragment 50-51. In someembodiments the method further comprises interrogating the biologicalsample for at least one indicium of a variant JCV VP1 capsid proteinthat comprises a deletion of amino acid fragment 54-55. In someembodiments the method further comprises interrogating the biologicalsample for at least one indicium of a variant JCV VP1 capsid proteinthat comprises a deletion of amino acid fragment 123-125. In someembodiments the method further comprises interrogating the biologicalsample for at least one indicium of a variant JCV VP1 capsid proteinthat comprises a deletion of amino acid fragment 125-134. In someembodiments the method further comprises interrogating the biologicalsample for at least one indicium of a variant JCV VP1 capsid proteinthat comprises a deletion of amino acid fragment 125-136.

In some embodiments the method further comprises interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein that comprises a deletion of one or more of amino acid fragments50-51, 54-55 and 123-125. In some embodiments the sample is interrogatedusing an assay capable of detecting at least one indicium of each ofvariant JCV VP1 capsid proteins having a deletion of at least one ofamino acid fragments 50-51, 54-55 and 123-125, and wherein the subjectis determined to have an increased susceptibility for PML if the samplecomprises at least one indicium of at least one of the variant JCV VP1capsid proteins.

In some embodiments the method further comprises interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein suspected of having low sialic acid binding.

In some embodiments the method further comprises interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein that comprises a substitution of at least one of amino acidresidues 55, 60, 265, 267, and 269. In some embodiments the sample isinterrogated using an assay capable of detecting at least one indiciumof each of variant JCV VP1 capsid proteins having a substitution of atleast one of amino acid residues 55, 60, 265, 267, and 269, and whereinthe subject is determined to have an increased susceptibility for PML ifthe sample comprises at least one indicium of at least one of thevariant JCV VP1 capsid proteins.

In some embodiments the biological sample is a blood sample. In someembodiments the biological sample is a CSF sample. In some embodimentsthe biological sample is a urine sample.

In some embodiments the subject is known to have been previouslyinfected with a wild-type JCV. In some embodiments a new biologicalsample from the subject is interrogated for at least one indicium of atleast one variant JCV VP1 capsid protein at least twice each year. Insome embodiments a new biological sample from the subject isinterrogated for at least one indicium of at least one variant JCV VP1capsid protein at least daily, weekly, monthly, bimonthly, quarterly,twice each year, yearly, every two years or every five years or anyfrequency in between.

In some embodiments the detection of at least one (e.g., 2, 3, 4, 5, ormore) indicium of a variant JCV VP1 capsid protein is used to identifythe subject as inappropriate for an immunosuppressive treatment. In someembodiments the detection of at least one, at least two, at least three,at least four, at least five, at least six, at least seven, at least 8,at least 9, at least 10 or more indicia of a variant JCV VP1 capsidprotein is used to identify the subject as inappropriate for animmunosuppressive treatment.

In some embodiments the detection of at least one indicium of a variantJCV VP1 capsid protein is used to recommend a modification of animmunosuppressive treatment for the subject. In some embodiments thedetection of at least one at least two, at least three, at least four,at least five, at least six, at least seven, at least 8, at least 9, atleast 10 or more indicia of a variant JCV VP1 capsid protein is used torecommend a modification of an immunosuppressive treatment for thesubject.

In some embodiments the absence of indicia of a variant JCV VP1 capsidprotein is used to identify the subject as appropriate for animmunosuppressive treatment.

In some embodiments the absence of indicia of a variant JCV VP1 capsidprotein is used to identify the subject as appropriate for continuedimmunosuppressive treatment.

In some embodiments the biological sample is interrogated for thepresence of an antibody that is specific for a variant JCV VP1 capsidprotein.

In some embodiments the biological sample is interrogated for thepresence of a variant JCV VP1 capsid protein.

In some embodiments the biological sample is interrogated for thepresence of a variant JCV VP1 capsid protein.

In some embodiments the biological sample is interrogated for thepresence of a nucleic acid sequence that encodes a variant JCV VP1capsid protein.

In some embodiments the biological sample is interrogated using an ELISAbased analysis.

Accordingly, in some embodiments, subjects can be screened to determinewhether they are at risk of developing PML. In certain embodiments,subjects can be screened to detect early stage PML before the diseaseprogresses to a serious clinical condition. A subject may be assayed forPML risk or early stage PML by interrogating a biological sampleobtained from the subject for indicia of exposure to a PML-associatedvariant of JCV, for example a JCV variant that is predicted to havereduced binding to sialic acid. The invention provides specificpositions in the major JC virus capsid protein (JCV-VP1) that areassociated with PML risk and/or PML disease progression. In someembodiments, the risk profile of a subject for PML is determined basedon the mutational status at one or more selected positions of the VP1protein of a JC virus to which the subject has been exposed.

Aspects of the invention are useful for detecting PML risk or earlystage disease progression in susceptible subjects, for example, inpatients that are immuno-compromised and/or being treated withimmuno-suppressive agents.

Accordingly, in one aspect the invention provides methods fordetermining that a subject is susceptible to PML. In some embodiments,the method comprises determining that a subject is susceptible to PML ifthe subject harbors a JCV variant suspected of having low sialic acidbinding properties (e.g., avidity or affinity).

In some embodiments, the method comprises determining whether a subjectharbors a JCV variant suspected of having low sialic acid binding, andidentifying the subject as being susceptible to PML if the subjectharbors the JCV variant. In some embodiments, the method comprisestesting a subject for the presence of a JCV variant suspected of havinglow sialic acid binding and identifying the subject as susceptible toPML if the presence of a JCV variant suspected of having low sialic acidbinding is detected.

In one aspect the invention provides methods for determining that asubject is appropriate for an immunosuppressive treatment. In someembodiments, a method comprises determining that a subject isappropriate for an immunosuppressive treatment if the subject does notharbor a JCV variant suspected of having low sialic acid binding. Insome embodiments, a method comprises determining whether a subjectharbors a JCV variant suspected of having low sialic acid binding; andidentifying the subject as appropriate for an immunosuppressivetreatment if the subject does not harbor the JCV variant, or identifyingthe subject as being inappropriate for an immunosuppressive treatment ifthe subject harbors the JCV variant.

In one aspect the invention provides methods for determining that asubject is inappropriate for an immunosuppressive treatment. In someembodiments, a method comprises determining that a subject isinappropriate for an immunosuppressive treatment if the subject harborsa JCV variant suspected of having low sialic acid binding.

In some of the embodiments of the methods provided herein determiningcomprises assaying a biological sample from the subject for an indiciumof the JCV variant suspected of having low sialic acid binding. In someof the embodiments of the methods provided herein a biological sample isa urine sample, a blood sample, plasma, serum, mucosal swab, or a CSFsample. In some of the embodiments of the methods provided herein theindicium is the presence of an antibody that can specifically bind to aJCV variant suspected of having low sialic acid binding. In some of theembodiments of the methods provided herein the indicium is the presenceof a nucleic acid sequence associated with a JCV polypeptide variantsuspected of having low sialic acid binding, wherein the nucleic acidsequence is not present in a wild type JCV.

In some of the embodiments of the methods provided herein the assayingcomprises contacting the biological sample with a JCV antibody andevaluating the sample for the presence of a JCV variant or JCV peptidevariant. In some of the embodiments of the methods provided herein theassaying comprises contacting the biological sample with a JCV peptideand evaluating the sample for the presence of a JCV antibody. In some ofthe embodiments of the methods provided herein the assaying comprisesperforming a PCR reaction on the biological sample and evaluating thesample for the presence of a JCV nucleic acid variant.

In some of the embodiments of the methods provided herein theimmunosuppressive treatment comprises administering an immunosuppressantdrug. In some of the embodiments of the methods provided herein theimmunosuppressive treatment comprises administration of an anti-VLA4antibody. In some of the embodiments of the methods provided herein, theimmunosuppressant drug is natalizumab.

In some of the embodiments of the methods provided herein the JCVvariant suspected of having low sialic acid binding is a JCV variantcomprising one or more mutations in the JCV sialic acid binding site. Insome of the embodiments of the methods provided herein the mutation inthe JCV sialic acid binding site is L55F, K60M, K60E, K60N, N265D,N265T, S267F, S267L, S269F, S269Y or S269C.

In some of the embodiments of the methods provided herein the low sialicacid binding of the JCV variant is lower than the sialic binding of WTJCV.

In some of the embodiments of the methods provided herein the subject isin need of treatment with an immunosuppressant drug or the subject isbeing treated with an immunosuppressant drug.

In one aspect the invention provides a method comprising obtaining abiological sample from a subject, assaying the biological sample for anindicium of a JCV variant suspected of having low sialic acid binding,wherein the subject is identified as being susceptible to PML if thebiological sample contains an indicium of the JCV variant.

In one aspect the invention provides a method comprising obtaining abiological sample from a subject, assaying the biological sample for anindicium of a JCV variant suspected of having low sialic acid binding,wherein the subject is identified as either i) appropriate for animmunosuppressive treatment if the biological sample does not containthe indicium of the JCV variant ii) inappropriate for animmunosuppressive treatment if the biological sample contains theindicium of the JCV variant.

In one aspect the invention provides a method comprising monitoring asubject receiving an immunosuppressive treatment for harboring a JCVvariant suspected of having low sialic acid binding.

In one aspect the invention provides a method comprising monitoring asubject receiving an immunosuppressive treatment for a sign of exposureto JCV, and, if the sign of exposure to JCV is detected, then monitoringthe subject for harboring a JCV variant suspected of having low sialicacid binding.

In some of the embodiments of the methods provided herein the monitoringcomprises periodically obtaining and assaying a biological sample fromthe subject for an indicium of the JCV variant.

In one aspect the invention provides a method comprising obtaining abiological sample from a subject, assaying the biological sample for anindicium of JCV, wherein if the biological sample contains an indiciumof JCV, the subject is periodically monitored for the presence of a JCVvariant suspected of having low sialic acid binding, and wherein if theclinical sample does not contain JCV, the subject is periodicallymonitored for an exposure JCV.

In one aspect the invention provides a method of determining whether atreatment regimen for administering an immunosuppressive agent to asubject should be modified, the method comprising obtaining a biologicalsample from a subject, assaying the biological sample for an indicium ofa JCV variant suspected of having low sialic acid binding, wherein thetreatment regimen should be modified for the subject if the biologicalsample has the indicium of the JCV variant.

In some of the embodiments of the methods provided herein the treatmentregimen should be modified by administering a lower dose of theimmunosuppressive agent, replacing the immunosuppressive agent with adifferent immunosuppressive agent, or halting administration of theimmunosuppressive agent.

In one aspect the invention provides a method comprising administering afirst immunosuppressant drug to a subject, and monitoring if the subjectharbors a JCV variant suspected of having low sialic acid binding. Insome of the embodiments of the methods provided herein the dosage and/orfrequency of administration of the first immunosuppressant drug isreduced if the subject harbors the JCV variant. In some of theembodiments of the methods provided herein the first immunosuppressantdrug is replaced with a second immunosuppressant drug if the subjectharbors the JCV variant. In some of the embodiments of the methodsprovided herein the subject is screened for a symptom of PML if thesubject harbors the JCV variant. In some of the embodiments of themethods provided herein the subject is treated for PML if the subjectharbors the JCV variant.

In one aspect the invention provides a method of detecting a JCV variantsuspected of having low sialic acid binding, the method comprisinginterrogating a biological sample from a subject using a highsensitivity assay specific for the indicium of a JCV variant suspectedof having low sialic acid binding. In some of the embodiments of themethods provided herein the interrogating comprises contacting thebiological sample with an antibody that can specifically bind the JCVvariant. In some of the embodiments of the methods provided herein theinterrogating comprises contacting the biological sample with a JCVvariant polypeptide.

In some of the embodiments of the methods provided herein theinterrogating comprises contacting the biological sample with a JCVvariant nucleic acid.

Aspects of the invention include panels of JCV-VP1 amino acid positionsat which sequence variations are associated with PML risk and/or diseaseprogression. Methods and compositions are provided for screeningsubjects to identify individuals that have been exposed to a variant JCvirus having PML-associated sequence variations at one or more of theVP-1 amino acid positions in a panel.

Aspects of the invention can be used to diagnose PML, or to evaluateand/or monitor PML disease progression, regression, and/or status in asubject.

Certain aspects of the invention relate to evaluating therapeutictreatments for PML.

Other aspects of the invention relate to vaccines against PML-associatedJC virus variants.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus. In some embodiments the assay interrogates at least oneJCV-VP1 position selected from positions 69, 74, 75, 113, 117, 128, 134,158, 164, 223, 271, 321, 332, and 345 in Table 1A for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus. In some embodiments the assay interrogates one positionfor the presence of a sequence variation. In some embodiments theposition is position 164. In some embodiments the subject is identifiedas being at risk for, or having, PML if a sequence variation is presentat position 164.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least eight JCV-VP1positions selected from the positions in Table 1A for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least eight JCV-VP1positions selected from the positions in Table 1A for the presence of asequence variation, wherein a sequence variation is one of the following55F, 60M, 60E, 61L, 66H, 66N, 69D, 74S, 75R, 113L, 117S, 123C, 128A,134G, 158L, 164K, 223A, 265D, 265T, 267F, 267L, 269F, 269Y, 271H, 321V,332E or 345K, and wherein the subject is identified as being at riskfor, or having, PML if a sequence variation is present at one or more ofthe interrogated positions.

In some embodiments, more than eight positions in Table 1A may beinterrogated. In some embodiments 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21 or 22 positions are interrogated.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least five JCV-VP1positions selected from the positions in Table 1B for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In some embodiments, more than five positions in Table 1B may beinterrogated. In some embodiments six or seven positions areinterrogated.

In one aspect, the invention provides a method for determining if asubject is at risk, or has, for progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least four JCV-VP1positions selected from the positions in Table 1C for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least two JCV-VP1positions selected from the positions in Table 1D for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In some embodiments, more than two positions in Table 1D may beinterrogated. In some embodiments 3, 4 or 5 positions are interrogated.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least two JCV-VP1positions selected from the positions in Table 1F for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In some embodiments, more than two positions in Table 1F may beinterrogated. In some embodiments 3 or 4 positions are interrogated.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising: assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least eight JCV-VP1positions selected from the positions in Table 1G for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if a sequence variation is present at one ormore of the interrogated positions.

In some embodiments, more than eight positions in Table 1G may beinterrogated. In some embodiments 9, 10, 11, 12, 13, 14 or 15 positionsare interrogated.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising: assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least one JCV-VP1position selected from the positions in Table 1H for the presence of asequence variation, and wherein the subject is identified as being atlow risk for, or not having, PML if a sequence variation is present atone or more of the interrogated positions. In some embodiments twopositions in Table 1H are interrogated.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising: assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least one JCV-VP1position selected from the positions in Table 1H for the presence of asequence variation, wherein a sequence variation is one of the following115E or 277K, and wherein the subject is identified as being at low riskfor, or not having, PML if a sequence variation is present at one ormore of the interrogated positions.

In some embodiments, two positions in Table 1H are interrogated. In oneaspect, the invention provides a method for determining if a subject isat risk for, or has, progressive multifocal leukoencephalopathy (PML),the method comprising assaying a biological sample from a subject for anindicium of exposure to a variant JC polyomavirus, wherein the assayinterrogates positions in selected regions of JCV-VP1 for the presenceof a sequence variation, and wherein the subject is identified as beingat low risk for, or not having, PML if less than a selected number ofsequence variations are present at the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75and 265-271 of JCV-VP1 for the presence of a sequence variation, andwherein the subject is identified as being at low risk for, or nothaving, PML if less than two sequence variations are present at theinterrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75,113-164, 223, 265-277 and 321-345 of JCV-VP1 for the presence of asequence variation, and wherein the subject is identified as being atlow risk for, or not having, PML if less than three sequence variationsare present at the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75,113-164, 223, 265-277 and 321-345 of JCV-VP1 for the presence of asequence variation, and wherein the subject is identified as being atlow risk for, or not having, PML if less than two sequence variationsare present at the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates position 164 and at leastone other JCV-VP1 position selected from the positions in Table 1A forthe presence of a sequence variation, and wherein the subject isidentified as being at risk for, or having, PML if a sequence variationis present at one or more of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates position 164 and at leastone other JCV-VP1 position selected from the positions in Table 1A forthe presence of a sequence variation, and wherein the subject isidentified as being at risk for, or having, PML if a sequence variationis present at two or more of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates positions in selectedregions of JCV-VP1 for the presence of a sequence variation, and whereinthe subject is identified as being at risk for, or having, PML if thenumber of amino acids with a specific characteristic present at theinterrogated positions has increased. In some embodiments, the specificcharacteristic is non-polarity.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75and 265-271 of JCV-VP1 for the presence of a sequence variation, andwherein the subject is identified as being at risk for, or having, PMLif the total number of non-polar amino acid variants (Gly, Ala, Val,Leu, Ile, or Pro) or aromatic variants (Phe, Tyr, or Trp) at theinterrogated positions has increased.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75,113-164, 223, 265-277 and 321-345 of JCV-VP1 for the presence of avariant, and wherein the subject is identified as being at risk for, orhaving, PML if the total number of non-polar amino acid variants oraromatic variants at the interrogated positions has increased.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75and 265-271 of JCV-VP1 for the presence of a sequence variation, andwherein the subject is identified as being at risk for, or having, PMLif the one or more of the sequence variations at the interrogatedpositions result in an increase in non-polar surface area of 10 squareangstrom or more per sequence variation.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates positions in selectedregions of JCV-VP1 for the presence of a sequence variation, and whereinthe subject is identified as being at low risk for, or not having, PMLif the number of amino acids with a specific characteristic present atthe interrogated positions has decreased. In some embodiments thespecific characteristic is polarity.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75and 265-271 of JCV-VP1 for the presence of a sequence variation, andwherein the subject is identified as being at risk for, or having, PMLif the total number of polar amino acid variants (Ser, Thr, Cys, Met,Asn, or Gln), positively charged variants (Lys, Arg, or His), ornegatively charged variants (Asp or Glu) at the interrogated positionshas decreased.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75,113-164, 223, 265-277 and 321-345 of JCV-VP1 for the presence of asequence variation, and wherein the subject is identified as being atrisk for, or having, PML if the total number of polar amino acidvariants, positively charged variants, or negatively charged variants atthe interrogated positions has decreased.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least positions 55-75and 265-271 of JCV-VP1 for the presence of a sequence variation, andwherein the subject is identified as being at risk for, or having, PMLif the one or more of the sequence variations at the interrogatedpositions result in a decrease in polar surface area of 10 squareangstrom or more per sequence variation.

In one aspect, the invention provides methods for interrogating selectedJCV-VP1 positions for sequence variations in a biological sample.

In some embodiments, interrogating selected JCV-VP1 positions forsequence variations comprises determining the presence in a biologicalsample of one or more antibodies that specifically bind polypeptidescomprising one or more amino acid sequence variations selected from thepanel of Table 1A or 1H. In some embodiments the presence of the one ormore antibodies is determined by specific binding to recombinantlyproduced polypeptides comprising the one or more amino acid sequencevariations. In some embodiments the presence of the one or moreantibodies is determined by specific binding to synthetically producedpolypeptides comprising the one or more amino acid sequence variations.

In some embodiments, interrogating selected JCV-VP1 positions forsequence variations comprises determining the presence in the biologicalsample of one or more polypeptides comprising the one or more amino acidsequence variations selected from the panel of Table 1A or 1H. In someembodiments the presence of the one or more polypeptides is detected byspecific binding of the polypeptides to one or more peptide bindingagents. In some embodiments the peptide binding agent is an antibody.

In some embodiments, interrogating selected JCV-VP1 positions forsequence variations comprises determining the presence in a biologicalsample of nucleic acids that code for the one or more amino acidsequence variations selected from the panel of Table 1A or 1H.

In some embodiments, interrogating selected JCV-VP1 positions forsequence variations is performed in a sample of blood, cerebrospinalfluid, serum, urine, sputum, bone marrow, brain, spleen or kidney, orother tissue.

In some embodiments, the method for determining if a subject is at riskfor, or has, progressive multifocal leukoencephalopathy (PML), comprisesassaying a biological sample from a subject for an indicium of exposureto a variant JC polyomavirus, wherein the exposure to variant JCVcomprises a current infection with a variant JCV.

In some embodiments, the method for determining if a subject is at riskfor, or has, progressive multifocal leukoencephalopathy (PML), comprisesassaying a biological sample from a subject for an indicium of exposureto a variant JC polyomavirus, the exposure to variant JCV comprises aprior infection, or the variant JCV is not detectable in the blood orurine of the subject.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least two JCV-VP1positions selected from the positions in Table 1F for the presence of asequence variation, and wherein the subject is identified as being atlow risk for, or not having, PML if no sequence variation is present atone of the interrogated positions.

In one aspect, the invention provides a method for determining if asubject is at risk for, or has, progressive multifocalleukoencephalopathy (PML), the method comprising assaying a biologicalsample from a subject for an indicium of exposure to a variant JCpolyomavirus, wherein the assay interrogates at least two JCV-VP1positions selected from the positions in Table 1G for the presence of asequence variation, and wherein the subject is identified as being atlow risk for, or not having, PML if no sequence variation is present atone of the interrogated positions.

In one aspect, the invention provides kits for diagnosing if a subjectis at risk for PML.

In some embodiments the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1A, Table 17 and/or Table 18, and/orone or more other variants described herein, and the kit furthercomprising instructions for using the polypeptide to determine thepresence of antibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1B, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1C, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1D, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1F, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1G, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1H, and the kit further comprisinginstructions for using the polypeptide to determine the presence ofantibodies that specifically bind these polypeptides.

In some embodiments, the kit for diagnosing if a subject is at risk forPML comprises one or more containers, each container containing apolypeptide comprising an amino acid sequence variations at position164, and the kit further comprising instructions for using thepolypeptide to determine the presence of antibodies that specificallybind these polypeptides.

In one aspect, the invention provides methods for determining onset,progression, or regression, of PML in a subject.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining thenumber of amino acid sequence variations in variant JCV-VP1 in a firstbiological sample obtained from the subject, determining the number ofamino acid sequence variations in variant JCV-VP1 in a second biologicalsample obtained from the subject at a later time than the first samplewas obtained, comparing the number of sequence variations in the firstsample with number of sequence variations in the second sample, whereina lower number of sequence variations in the first sample compared withthe number of sequence variations in the second sample indicates onsetor progression of PML in the subject, and wherein a higher number ofsequence variations in the first sample compared with the number ofsequence variations in the second sample indicates regression of PML inthe subject.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1A.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1B.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1C.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1D.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1F.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are selectedfrom the positions in Table 1G.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the sequence variations are determinedby comparing the variant JCV-VP1 to a wild type JCV-VP1.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the method comprises determining if asequence variation is present at position 164 in variant JCV-VP1 in afirst biological sample obtained from the subject, determining if asequence variation is present at position 164 in variant JCV-VP1 in asecond biological sample obtained from the subject at a later time thanthe first sample was obtained, wherein absence of the sequence variationin the first sample and the presence of the sequence variation in thesecond sample indicates onset or progression of PML in the subject, andwherein presence of the sequence variation in the first sample andabsence of the sequence variation in the second sample indicatesregression of PML in the subject.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method further comprises additionalmonitoring of the onset, progression, or regression, of PML in a subjectby obtaining additional samples from the subject at later time points,determining the number of amino acid sequence variations in JCV-VP1 inthese samples, and comparing the number of sequence variations in thesesamples to the number of sequence variations in one or more previoussamples.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the subject is monitored whileundergoing treatment with an immunosuppressant. In some embodiments theimmunosuppressant is natalizumab.

In one embodiment of the method for determining onset, progression, orregression, of PML in a subject, the method comprises determining theviral load of a variant JCV-VP1 in a first biological sample obtainedfrom the subject, determining the viral load of a variant JCV-VP1 in asecond biological sample obtained from the subject at a later time thanthe first sample was obtained, comparing the viral load in the firstsample with viral load in the second sample, wherein a lower viral loadin the first sample compared with the viral load in the second sampleindicates onset or progression of PML in the subject, and wherein ahigher viral load in the first sample compared with the viral load inthe second sample indicates regression of PML in the subject.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1A.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1B.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1C.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1D.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1F.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises one ormore sequence variations selected from the positions in Table 1G.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the variant JCV-VP1 comprises asequence variation at position 164.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the viral load of variant JCV-VP1 iscompared to the viral load of a wild type JCV-VP1.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the method further comprises additionalmonitoring of the onset, progression, or regression, of PML in a subjectby obtaining additional samples from the subject at later time points,determining the viral load of variant JCV-VP1 in these samples, andcomparing the viral load in these samples to the viral load in one ormore previous samples.

In some embodiments of the method for determining onset, progression, orregression, of PML in a subject, the method comprising determining theviral load of a variant JCV-VP1, the subject is monitored whileundergoing treatment with an immunosuppressant. In some embodiments theimmunosuppressant is natalizumab.

In one aspect, the invention provides methods for monitoring response totreatment for PML in a subject.

In some embodiments of the method for monitoring response to treatmentfor PML in a subject, the method comprises determining the number ofamino acid sequence variations in JCV-VP1 in a first biological sampleobtained from the subject, administering the PML treatment to thesubject, determining the number of amino acid sequence variations inJCV-VP1 in a second sample, wherein the second sample is obtained fromthe subject after treatment and at a time later than the first sample,and comparing the number of amino acid sequence variations in the firstsample with the number of amino acid sequence variations in the secondsample, wherein a lower number of amino acid sequence variations in thesecond sample than in the first sample indicates that the subject isresponsive to the PML treatment.

In some embodiments of the method for monitoring response to treatmentfor PML in a subject, the method comprises determining the viral load ofa JCV variant in a first biological sample obtained from the subject,administering the PML treatment to the subject, determining the viralload of a JCV variant in a second sample, wherein the second sample isobtained from the subject after treatment and at a time later than thefirst sample, and comparing the viral load in the first sample with theviral load in the second sample, wherein a lower viral load in thesecond sample than in the first sample indicates that the subject isresponsive to the PML treatment.

In one aspect, the invention provides methods for selecting a course oftreatment of a subject having or suspected of having PML.

In some embodiments of the method for selecting a course of treatment ofa subject having or suspected of having PML, the method comprisesdetermining the number of amino acid sequence variations in JCV-VP1 in abiological sample obtained from the subject, comparing the number ofsequence variations to a control number of sequence variations,determining the stage of PML based at least in part on the difference inthe number of sequence variations in the sample compared to the controlnumber of sequence variations, and selecting a course of treatment forthe subject appropriate to the stage of PML of the subject.

In some embodiments of the method for selecting a course of treatment ofa subject having or suspected of having PML, the method comprisesdetermining the viral load of a JCV variant in a biological sampleobtained from the subject, comparing the viral load to the viral load ina control sample, determining the stage of PML based at least in part onthe difference in viral load in the sample compared to the controlsample, and selecting a course of treatment for the subject appropriateto the stage of PML of the subject.

In one aspect, the invention provides methods for deciding or aiding inthe decision to discontinue treatment of a subject with one or moreimmunosuppressants.

In some embodiments of the method for deciding or aiding in the decisionto discontinue treatment of a subject with one or moreimmunosuppressants comprises determining the number of amino acidsequence variations in JCV-VP1 in a biological sample obtained from thesubject, comparing the number of sequence variations to a control numberof sequence variations, determining a decision to discontinue treatmentof a subject with one or more immunosuppressants based at least in parton the difference in the number of sequence variations in the samplecompared to the control number of sequence variations.

In some embodiments of the method for deciding or aiding in the decisionto discontinue treatment of a subject with one or moreimmunosuppressants comprises determining the vial load of a JCV variantin a biological sample obtained from the subject, comparing the viralload to a viral load in a control sample, determining a decision todiscontinue treatment of a subject with one or more immunosuppressantsbased at least in part on the difference in viral load in the samplecompared to the control sample.

In some embodiments of the method for deciding or aiding in the decisionto discontinue treatment of a subject with one or moreimmunosuppressants, the immunosuppressant is natalizumab.

In one aspect, the invention provides methods for identifying candidatetherapeutic compounds.

In some embodiments of the method for identifying a candidatetherapeutic compound, the method comprises contacting an isolatedpolypeptide comprising one or more amino acid sequence variationsselected from the panel of Table 1A, Table 17 and/or Table 18, and/orone or more other variants described herein, with a compound todetermine if the compound binds to the isolated polypeptide, wherein ifthe compound binds to the isolated polypeptide the compound is acandidate therapeutic compound.

In one aspect, the invention also provides candidate therapeuticcompounds identified by contacting an isolated polypeptide comprisingone or more amino acid sequence variations selected from the panel ofTable 1A, Table 17 and/or Table 18, and/or one or more other variantsdescribed herein.

In some embodiments of the method for identifying a candidatetherapeutic compound, the method comprises determining a number of aminoacid sequence variations in JCV-VP1 in a first biological sampleobtained from the subject, administering a compound, determining thenumber of amino acid sequence variations in JCV-VP1 in a secondbiological sample obtained from the subject at a time afteradministration of the compound, wherein if the number of amino acidsequence variations in JCV-VP1 in the second sample is lower than in thefirst sample the compound is a candidate therapeutic compound.

In one aspect, the invention also provides candidate therapeuticcompounds identified by determining a number of amino acid sequencevariations in JCV-VP1 in a first biological sample obtained from thesubject, administering a compound, and determining the number of aminoacid sequence variations in JCV-VP1 in a second biological sampleobtained from the subject at a time after administration of thecompound.

In some embodiments of the method for identifying a candidatetherapeutic compound, the method comprises determining a viral load of aJCV variant in a first biological sample obtained from the subject,administering a compound, determining the viral load of a JCV variant ina second biological sample obtained from the subject at a time afteradministration of the compound, wherein if the viral load in the secondsample is lower than in the first sample the compound is a candidatetherapeutic compound.

In one aspect, the invention also provides candidate therapeuticcompounds identified by determining a viral load of a JCV variant in afirst biological sample obtained from the subject, administering acompound and determining the viral load of a JCV variant in a secondbiological sample obtained from the subject at a time afteradministration of the compound

In one aspect, the invention also provides vaccines comprising apolypeptide having a sequence variation at one or more JCV-VP1 positionsselected from the positions in Table 1A, Table 17 and/or Table 18,and/or one or more other variants described herein. In some embodimentsthe vaccine comprises two or more polypeptides having a sequencevariation at one or more JCV-VP1 positions selected from the positionsin Table 1A. In some embodiments, the vaccine further comprises anadjuvant.

In one aspect, the invention also provides methods of immunizing asubject against PML, the method comprising administering a vaccinecomprising one or more polypeptides having a sequence variation at oneor more JCV-VP1 positions selected from the positions in Table 1A, Table17 and/or Table 18, and/or one or more other variants described herein.

In one aspect, the invention also provides an isolated antibody thatspecifically binds to a polypeptide having a sequence variation at oneor more JCV-VP1 positions selected from the positions in Table 1A. Insome embodiments the polypeptide has 2, 3, 4, 5, or more sequencevariations at JCV-VP1 positions selected from the positions in Table 1A,Table 17 and/or Table 18, and/or one or more other variants describedherein. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments the antibody is a polyclonal antibody.

In certain embodiments, the antibody specifically binds to a VP1particle comprising at least one VP1 polypeptide containing a sequencevariation described herein. A VP1 particle useful in the invention canbe produced using methods known in the art, in general by expressing arecombinant VP1 polypeptide comprising a variant described herein. A VP1particle contains at least 2, 4, 10, 20, 30, 40, or 50 VP1 polypeptides.In some embodiments, the VP1 particle comprises only VP1 polypeptidescontaining a variant. In other embodiments, the VP1 particle is aheterogeneous particle containing more than one VP1 polypeptidesequence; e.g., more than one variant polypeptide or at least onevariant polypeptide and at least one wild type polypeptide.

The invention also encompasses an isolated nucleic acid sequenceencoding a VP1 polypeptide variant (e.g., containing one or morevariants described in Tables 1A-1H, Table 17 and/or Table 18, and/or oneor more other variants described herein) or a peptide of such apolypeptide, the polypeptide or peptide includes the sialic acid bindingregion of a VP1 polypeptide. In some cases, the invention includes suchan isolated nucleic acid sequence operatively linked to a heterologousexpression control sequence. The invention also includes vectors andhost cells, transiently or stably transfected containing such nucleicacid sequences. Methods for producing such sequences, vectors, and hostcells are known in the art.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the comparison of a reference JCV-VP1 sequence to the SV40COA sequence;

FIG. 2 shows a model of JCV-VP1;

FIG. 3 shows VP1 peptide sequences;

FIG. 4 shows a phylogenetic distribution of PML associated viruses;

FIG. 5 shows a structural model of JCVVP1/NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc tetrasaccharidecomplex;

FIG. 6 shows the quality of the viral like particles;

FIG. 7 shows that WT JCV binds to some glycans;

FIG. 8 shows the structure of selected gangliosides and their ability tobind to WT JCV;

FIG. 9 shows that the F55 and F269 mutations are not capable of bindingNeu5Acα(2-3) and α(2-6) glycans;

FIG. 10 shows the structure of selected gangliosides and their abilityto bind to mutant JCV;

FIG. 11 compares the ability of WT and mutant to JCV to bind selectedgangliosides;

FIG. 12 shows that mutant JVC is still capable of binding glial celllines;

FIG. 13 shows that the mutant specific JCV antibodies can bedistinguished from a WT JCV antibody;

FIG. 14 shows an assay for distinguishing mutant antibodies from WT;

FIG. 15 compares mutant JCV and WT JCV antibodies for their ability tobind to a mutant JCV-VLP;

FIG. 16 compares mutant JCV and WT JCV antibodies for their ability tobind to a mutant JCV-VLP;

FIG. 17 shows that a patient that has the F269 mutant virus hasdeveloped an antibody response against the WT virus but not against theF269 mutant virus;

FIG. 18 shows that (top) rabbit immunized with non-mutant VLP raisesantibody to the site (S269 in this cases) that could get mutated;

FIG. 19 shows that higher CSF JCV DNA levels were found in patients withmutations of the BC and HI loops than in those with mutations of the DEloop or no mutations;

FIG. 20 shows a structural model of JCVVP1/NeuNAc-(a2,3)-Gal-(b1,3)-[(a2,6)-NeuNAc]-Glc-NAc tetrasaccharidecomplex; and,

FIG. 21 shows PML specific mutations of VP1 abolish or drasticallychange specificity of viral capsid protein VP1 for sialatedgangliosides.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to identifying and managing thehealthcare of subjects who are at increased risk of progressivemultifocal leukoencephalopathy (PML). PML can be caused by a JCpolyomavirus (JCV) infection in humans, particularly in subjects thathave a weakened immune system. However, PML does not develop in allJCV-infected subjects having weakened immune systems. According to theinvention, only certain JCV variants cause PML. In some embodiments, JCVvariants that have reduced sialic acid binding are particularly likelyto cause PML. Accordingly, aspects of the invention relate to methodsand compositions for detecting the presence of JCV variants predicted tohave reduced binding to sialic acid.

According to aspects of the invention, and without wishing to be boundby theory, a JCV variant that has low sialic acid binding, relative tothe sialic binding of a wild-type JCV, has reduced binding to peripheralsites (e.g., sialic acid on peripheral cells, proteins, sugars, or othermolecules). Therefore, in some embodiments these variants are morelikely to spread to other parts of the body, for example, the CNS. Inaddition, according to aspects of the invention, subjects with healthyimmune systems control the spread of certain JCV variants with reducedsialic acid binding. However, in subjects with compromised immunesystems, these variants are more likely to evade the immune system andprogress to PML.

Accordingly, aspects of the invention relate to compositions and methodsfor detecting the presence of JCV variants that are predicted to havelow sialic acid binding (e.g., low binding or avidity) relative to thebinding of normal or wild-type JCV. In some embodiments, JCV variantswith one or more mutations in the sialic acid binding domain of a JCVcapsid protein are predicted to have low sialic acid binding properties.In some embodiments, specific variants described herein are predicted tohave low sialic acid binding properties.

According to aspects of the invention, subjects that harbor JCV variantswith one or more mutations that are predicted to reduce sialic acidbinding are identified as having increased susceptibility to PML (e.g.,compared to a subject that does not harbor JCV or such a JCV variant),particularly if their immune system is compromised. Accordingly,subjects that harbor a reduced sialic acid binding JCV variant are atrisk of developing PML if they are treated with an immunosuppressivedrug (e.g., natalizumab or other immunosuppressive drug).

In some embodiments, JCV mutations that are associated with PMLsusceptibility in a subject are mutations at positions 122, 2, and 66and deletions of amino acids 50-51, 123-125 and 126-134 in the VP1capsid protein of JCV. In some embodiments, JCV mutations that areassociated with PML susceptibility in a subject are H122R, A2V, andD66G. In some embodiments, JCV mutations that are associated with PMLsusceptibility in a subject are H122R, A2V, and D66G and 283I. In someembodiments a subject is susceptible to PML if the JCV VP1 capsidprotein comprises a substitution of at least one of amino acid residues122, 2, 66, 55, 60, 265, 267, and 269, or one or more deletions of aminoacids 50-51, 123-125 and 126-134.

Aspects of the invention are useful to assist in the selection and/ormonitoring of a therapy for a subject that is in need of animmunosuppressive treatment (e.g., a subject with multiple sclerosis, orany other condition that can be treated with one or moreimmunosuppressive drugs). In some embodiments, aspects of the inventioncan be used to identify subjects that are susceptible to PML prior toinitiating an immunosuppressive treatment. In some embodiments, subjectsthat are receiving an immunosuppressive treatment may be monitored forthe appearance of JCV variants that are associated with increased riskfor PML. In some embodiments, if a subject is identified as having a JCVvariant with reduced binding to sialic acid, then the subject is i) nottreated with an immunosuppressive drug, ii) treated with a low dosage orfrequency of drug administration, and/or iii) monitored regularly forearly symptoms of PML. If symptoms of PML appear during treatment, thetreatment may be halted or the amount or frequency of drugadministration may be reduced. In some embodiments, an alternativeimmunosuppressive drug may be substituted for a first drug if one ormore symptoms of PML are present.

Aspects of the invention may be used to implement patient monitoringprocedures. In some embodiments, patients are first assayed to determinewhether they have been exposed to JCV (e.g., by assaying patient serumfor the presence of a JCV antibody). Patients that have not been exposedto JCV are identified as having a low risk for PML. However, in someembodiments, such patients are monitored periodically to determinewhether or when they are exposed to JCV. If signs of JCV exposure areidentified in a subject prior to treatment or during treatment, then thesubject can be evaluated for the presence of one or more JCV variantsassociated with increased risk for PML (e.g., one or more variantspredicted to have reduced sialic acid binding relative to a normal orwild type form of JCV). Patients may be monitored periodically for thepresence of PML-associated JCV variants. If a patient is found to harborone or more PML-associated JCV variants, then the patient may bemonitored carefully for signs of PML, the patient's treatment may bestopped or altered, and/or the patient may be treated with one or moreprophylactic or therapeutic drugs to help protect the patient from PML.

In some embodiments a patient or subject is monitored daily, weekly,biweekly, monthly, bimonthly, quarterly, twice a year, yearly or eachtwo years. In some embodiments a patient or subject is monitored whenthe patient or subject is undergoing treatment with immunosuppressants.

According to aspects of the invention, a JCV variant may be predicted tohave low sialic acid binding (e.g., affinity or avidity) if the varianthas one or more mutations in the sialic binding pocket of the JVC VP1protein. In some embodiments, mutations in any one or more of the aminoacids that are within 12 Angstroms of a molecule bound in the sialicacid binding pocket can be predicted to affect sialic acid binding. Alist of VP1 amino acids within 12 Angstoms of a molecule bound in thesialic acid binding pocket is provided in Table 16 in Example 12.Accordingly, a JCV variant with a mutation in one or more of these aminoacids may be identified as being a candidate for reduced binding tosialic acid and therefore increased risk for causing PML.

According to aspects of the invention, several differentnaturally-occurring JCV VP1 sequences may be used as normal or wild-typereference sequences if they are from JCV variants that are notassociated with increased risk for PML as described herein. Examples ofsuch reference sequences are described in more detail herein and, forexample, in Cubitt et al., Predicted amino acid sequences for 100 JCVstrains, Journal of NeuroVirology, 7: 339-344, 2001, the sequencedisclosures of which are incorporated herein by reference in theirentirety.

According to aspects of the invention, the sialic acid bindingproperties (e.g., affinity or avidity) of a variant JCV may be measuredusing any suitable assay. For example, JCV and or JCV VP1 binding tosialic acid may measured using hemmaglutination assays, direct bindingto immobilized molecules (e.g., gangliosides, sugars, glycoproteins,etc.), cell-based binding assays (e.g., where binding is detected withlabeled antibodies using flow cytometry), etc., or any combinationthereof. In some embodiments, low binding is more than a 5 foldreduction in binding (e.g., avidity or affinity) relative to the bindingof a normal or wild-type JCV reference as described herein. However, insome embodiments, low binding is more than about 10 fold, 50 fold, 100fold, 200 fold, 500 fold, 1,000 fold, 5,000, 10,000 fold or greaterreduction in binding relative to a normal or wild-type JCV reference.

Accordingly, aspects of the invention relate to determining whether asubject is susceptible to PML due to the presence of a PML-associatedJCV variant. In some embodiments, this information can be used todetermine whether the subject is suitable for treatment with animmunosuppressive agent. In some embodiments, this information can beused to determine whether a subject being treated with animmunosuppressive agent should continue the treatment or should beswitched to a different dosage, regimen, and/or type ofimmunosuppressive drug. In certain embodiments, the detection of aPML-associated JCV variant in a subject being treated with animmunosuppressive agent provides a basis for discontinuing thetreatment, at least for a predetermined period of time.

Patients being treated with an immunosuppressive agent can be monitoredperiodically for the presence of a JCV infection. In some embodiments,if a subject is known or identified to have a JCV infection, the subjectmay be periodically monitored for the presence of a JCV variant that ispredicted to have a reduced binding for sialic acid.

Accordingly, the invention provides methods and compositions fordetermining if a subject is at risk for progressive multifocalleukoencephalopathy (PML) or has PML (e.g., early stage PML). In oneembodiment, a biological sample of a subject is interrogated forexposure to a variant JC virus having one or more sequence variationsassociated with PML, for example, one or more sequence variantspredicted to result in reduced sialic acid binding. In some embodimentsa plurality of specific predetermined VP-1 positions are interrogatedfor their mutational status. The pattern of sequence variations found atthe specific positions provides a risk profile of the subject for PML.

In some embodiments, the invention provides panels of JCV-VP1 amino acidpositions at which sequence variations are associated with PML riskand/or PML disease progression. Methods and compositions are providedfor assaying biological samples for indicia of PML risk. In someembodiments, JC virus polypeptides and/or nucleic acids may be isolatedand analyzed to determine whether they have sequence variations at oneor more predetermined VP-1 positions. However, in certain circumstances,JC virus can be difficult to isolate from certain biological samplesobtained from subjects that have low JC viral loads even through theyhave been exposed to JC virus and may have a current JC viral infection.Nonetheless, a subject's exposure to a PML-associated variant JC viruscan be inferred from the presence, in a biological sample obtained fromthe subject, of antibodies (e.g., serum antibodies) against one or morePML-associated variant JCV-VP1 polypeptides.

JCV sequences in the brain of PML patients are so variable thatidentical JCV sequences have never been detected in different PMLpatients (Yogo & Sugimoto, 2001). However, aspects of the invention arebased on an analysis of JCV sequences from healthy and PML subjects andthe identification of a subset of positions in the VP1 sequence at whichamino acid substitutions are strongly correlated with PML. In oneaspect, the invention provides guidance as to which positions in JCV-VP1need to be interrogated to arrive at a risk profile for PML. In oneaspect, the invention provides novel panels of VP1 amino acid positionsat which sequence variations are predictive of a risk of PML in asubject infected with the corresponding variant JCV. Aspects of theinvention relate to screening subjects for the presence of signs orindicia of JCV variants with reduced binding to sialic acid. In someembodiments, panels of JCV sequence variations associated with differentPML risk profiles are provided in Table 1A-1H and described in moredetail herein.

JCV and VP1

In some embodiments, JCV sequence variations associated with PML arefound at selected positions in the major capsid protein (VP1) of JCV.Sequences of many VP1 proteins described in the literature have beenanalyzed to identify a subset of VP1 positions where sequence variationsare strongly associated with PML. According to the invention, an aminoacid is identified as mutant or variant at a specific position on VP-1if it differs from the amino acid at that position in a consensussequence or other reference sequence (e.g., the sequence of a wild typearchetype virus). In some embodiments the consensus sequence for the VP1protein of JCV from healthy subjects is provided by GenBank IDNo#37050849 (AAQ88264; shown as the VP1 sequence in FIG. 1). JCV-VP1 isconserved when compared to SV40 as shown in FIG. 1. A number ofdifferent archetypes of JCV have been identified. As used herein, anarchetype is a JCV found in the urine of healthy individuals. In someembodiments, the VP1 sequence of FIG. 1 is a reference sequence. In someembodiments, a reference sequence is one or more of the archetypes ofZheng et al. (2005). The three main archetypes are determined by region(EU archetype is found in people of European ancestry and the CY and MYarchetypes are found in people of Asian ancestry). While PML-associatedsequence variations were identified based on comparisons to a“consensus” sequence, several archetypes have the same amino acids atthe positions identified as PML-associated, and the invention cantherefore be practiced based on sequence comparisons to any of thearchetypal sequences. In other embodiments, assays can be based ondetermining whether a JCV has one or more predetermined amino acids atcertain positions without performing a comparison to a referencesequence. It should be appreciated that methods for determining thepresence of certain JCV-VP1 variant sequences can be based on a directcharacterization of JCV nucleic acids or polypeptides isolated from asubject. Alternatively, the presence of variant JCV in a subject (orprior exposure of the subject to variant JCV) can be inferred bydetecting subject antibodies (e.g., serum antibodies) that are specificfor one or more of the variant JCV-VP1 sequences. It should beappreciated that aspects of the invention also provide for comparing aJCV-VP1 sequence to other reference sequences. However, even if otherreference sequences are used, an analysis may involve determining theidentity of an amino acid at one or more of the PML associated VP1positions described herein.

According to the invention, the sequence analysis of VP1 variantsidentified “hot spot” regions of sequence variations associated withrisk for PML. In some embodiments, sequence variations that areindicative of risk for PML are located in the surface loops of the VP1protein. These positions can be found in Tables 1B-1D. In someembodiments, the total number of sequence variations in a specific areaof the VP1 protein may be indicative of risk for PML (See Table 2). Insome embodiments, the presence of variant amino acids at 2 or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) of the positions describedherein may be indicative of risk for PML. The polarity of these regionsmay be indicative of risk for PML and the risk for PML may increase ifpolar amino acids are replaced with non-polar amino acids. In someembodiments, a loss of a polar amino acid at any one or more of thepositions identified in Table 1A or Tables 1B-1D results in a JCV virushaving an increased association with PML. In some embodiments, a loss ofa polar amino acid at any one or more positions within one of thestructural regions shown in FIG. 2 (e.g., within a region defined bypositions 55-75 or 265-271 of the JCV-VP1 polypeptide sequence) resultsin a JC virus having an increased association with PML. Similarly, incertain embodiments a gain of a non-polar amino acid at any one or moreof the positions identified in Table 1A or Tables 1B-1D results in a JCVvirus having an increased association with PML. Also, in certainembodiments, a gain of a non-polar amino acid at any one or morepositions within one of the structural regions shown in FIG. 2 (e.g.,within a region defined by positions 55-75 or 265-271 of the JCV-VP1polypeptide sequence) results in a JC virus having an increasedassociation with PML. FIG. 2 shows a predicted JCV-VP1 structurehighlighting regions where certain amino acids positions identified asbeing associated with PML were mapped to the structure. The predictedstructure of the JCV-VP1 protein is based on the crystal structure ofthe SV40 coat protein 1 and the sequence similarity between the JCV andSV40 proteins. Parsing of the sequence variations showed that a decreasein the number of polar amino acids and/or an increase in the number ofnon-polar amino acids on the surface area of the VP1 protein resulted inan increased risk for PML (See Example 3). In one embodiment, a subjectis at risk for PML if a sequence variation in one or more of the surfaceloops results in an increase in non-polar surface area of 10-squareangstrom or more per sequence variation, or a decrease in polar surfacearea of 10-square angstrom or more per sequence variation. While thecurrent invention is not limited to a specific mechanism it is believedthat the changes in the surface loop may impede antibodies from bindingto the JCV-VP1 and/or facilitate the JCV VP1 interaction with itsreceptors such as carbohydrate and/or a protein receptor, such as 5HT2A.Changes in a surface loop of VP1 (e.g., an increase in non-polar aminoacids or surface area and/or a decrease in polar amino acids or surfacearea) may increase the affinity of VP1 for the cell receptor andfacilitate viral entry and/or infection of a host cell, or certainspecific host cells important for PML progression.

Panels of Sequence Variations

It should be appreciated that interrogating a position may comprisesboth determining the amino acid at that position and comparing thatamino acid to a reference amino acid (e.g., the amino acid found in thewild type/control variant, for example in the consensus sequence or anarchetype sequence).

It should be appreciated that any single variant or combination ofvariants can be indicative of risk for PML. It should be appreciatedthat the total number of variants present in a certain region or in aselected group of interrogated variants may indicate a risk for PML.

Aspects of the invention relate to providing one or more positions on aJCV-VP1 amino acid sequence that correspond to JCV variants that areassociated with risk for PML. For example, each of the positions shownin Table 1A are associated with risk for PML and any suitable assay canbe used to detect the presence of, or indicators (indicia) of pastexposure to, a JCV variant having an amino acid variant at one or moreof the selected positions. In some embodiments, an assay can be used todetect the presence of, or exposure to, a JCV variant having a variantamino acid variant at two or more positions selected from the positionslisted in Table 1A, wherein the presence of the variant/variants isindicative of PML.

In some embodiments, JCV mutations that are associated with PMLsusceptibility in a subject are mutations at positions 122, 2, and 66and deletions of amino acids 50-51, 123-125 and 126-134 in the VP1capsid protein of JCV. In some embodiments, JCV mutations that areassociated with PML susceptibility in a subject are H122R, A2V, andD66G. In some embodiments a subject is susceptible to PML if the JCV VP1capsid protein comprises a substitution of at least one of amino acidresidues 122, 2, 66, 55, 60, 265, 267, and 269, or one or more deletionsof amino acids 50-51, 123-125 and 126-134.

In some embodiments, the selected positions are interrogated bycomparing the amino acid present at that position to the amino acidpresent in a reference. If the amino acid present at a selected positiondiffers from the amino acid found in the reference, the position is saidto be mutated. Accordingly, in some aspects the invention providestables (e.g., databases) of containing lists of variants that arerelated to a risk for PML. The databases also provide variants andvariants that are protective of PML. In some embodiments any variantfound at a selected position is indicative of risk for PML. In furtherembodiments the tables provide groups of variants associated with PMLthat can be interrogated as a group to arrive at a risk profile for PMLfor a subject. It should be appreciated that many of the variantsidentified in the reference database are not very common. For instance avariant indicative of risk for PML may be found in only 10% of thesamples of a person known to be at risk for PML. To arrive at a riskprofile for a subject more than one position may need to beinterrogated. Accordingly, in one aspect the invention provides tablesof groups of positions and number of positions within that group thatneed to be identified to arrive at a risk profile for PML. In someembodiments, at least 8 (e.g., 8, 9, 10 or more, or all) positions inTable 1A need to be interrogated to be arrive at a subject's riskprofile for PML. Similarly, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more or allpositions in any of the tables may be interrogated.

Amino acids found at the positions indicated in the Tables may beindicative of risk for PML by comparing the amino acid to an amino acidfound in a reference. For instance, in Table 1A, the wild type (e.g.,reference/consensus) amino acid at position 55 is leucine (L). In someembodiments, any amino acid found at position 55 that is not leucine isindicative of risk for PML. In some embodiments, a particular variant isassociated with risk for PML. For instance, the presence of aphenylalanine (F) at position 55 may be indicative of risk for PML.Accordingly, the emergence of a variant amino acid (e.g., Phe) atposition 55 may be indicative of onset or progression of PML. Similarly,the emergence of one or more other variant amino acids described hereinfor PML-associated positions on VP1 may be indicative of onset orprogression of PML. In some embodiments, the presence of an amino acidwith a particular characteristic may be indicative of the risk for PML.For instance, the presence in the JCV variant of an amino acid that isless polar than the amino acid found in the reference sequence isindicative of risk for PML. In addition to being indicative for risk forPML, the variants found at specific positions may also be used todetermine onset, progression or regression in a subject. In someembodiments, the variants found at specific positions allow for themonitoring of treatment of a subject for PML. In some embodiments, thevariants found at specific positions allow for the selection of atreatment of a subject having or suspected of having PML.

In some embodiments, amino acids found at specific amino positions canbe indicative as being protective for PML, e.g., identifying the personfor being at lower risk for developing PML.

It should be appreciated that in some embodiments, the risk for PML canbe determined without comparing the amino acid determined at aparticular position to a reference sequence. For instance, if in asample position 55 is interrogated and the amino acid found at thatposition is a phenylalanine, then the subject is determined to be atrisk for PML. This determination can be made without comparing the aminoacid found at position 55 to a wild type reference.

It should be appreciated that the invention provides methods fordetermining both the current and past existence of JCV variants. Asubject that has been exposed to a JCV variant, but that currently cannot be identified as being infected with that JCV variant, can bedetermined to be exposed to that JCV variant by determining the presenceof indicators for that JCV variant. A subject can develop antibodiesagainst any foreign agent, including viruses and bacteria, to which thesubject is exposed. The presence of a virus, or variant of the virus ina subject can therefore be diagnosed by the detection of an antibodyspecific to that virus or virus variant. If the virus replicates andmutates (and thus creates a new variant) the body will develop newantibodies specific for the mutated version of the virus. Even if aspecific virus variant is no longer present in the body, past exposureto the variant can still be determined by the presence of the antibody.Accordingly, in one embodiment the mutational status of the JCV variants(both current and past exposure) is determined by the detection of thepresence of antibodies against one or more specific variants. In oneembodiment, the presence of a VP1-antibody is determined by specificbinding of the antibody to one or more polypeptides comprising one ormore of the variants. It should be appreciated that antibodies can bedetermine qualitatively and quantitatively. An antibody detection assayof the invention therefore facilitates the determination of all viralvariants the subject has ever been exposed to and an estimate of theviral load of variants currently present in the body through thequantization of the specific antibodies.

In one embodiment, the amino acid variants are detected directly, e.g.,the presence of virus variants in a subject is determined by determiningthe presence of polypeptides comprising one or more variants of thevariants. Agents are developed that can specifically bind a particularpolypeptide. In some embodiments these agents are antibodies. In someembodiments, the variants are determined by determining the sequence ofthe nucleic acids encoding the variants. A sample is obtained from asubject and analyzed for the presence of a particular virus variant. Itshould be appreciated that specific JCV variants may only be present inthe brain and spinal fluid, while “wild type” variants may be present inother tissues in the same subject. The presence of polypeptides may bedetermined by antibodies specific for these polypeptides. The assay willallow for the determination of both the presence of specific variantsand the quantitation of these variants (the viral load), in addition theratio of variant to wild type can be determined.

It should be appreciated that an immunosupressive agent may increase thesusceptibility of a subject to the progression or flare up of a latentmicrobial infection or to the contraction of a new microbial infection.In some embodiments the microbial infection is infection by JCV, whichcaused PML. Accordingly, the risk for PML may be assessed prior toinitiating treatment with an immunosuppressive agent, duringadministration of an immunosupressive agent, or assessed after animmunosupressive agent has been administered or after treatment withimmunosupressive treatment has been terminated.

In some embodiments, the risk for PML as determined by the methods ofthe invention will aid in deciding on treatment with immunosuppressantsor immunosuppresive agents. In some embodiments an increased risk forPML diagnosed during treatment with immunossuppressents may suggest amodification or termination of the treatment regimen withimmunosuppressants.

TABLE 1A Panel of JCV-VP1 variants PML patients PML PML Healthy (brain +patients patients JCV individ- peripheral (peripheral (brain VP1 ualstissue) tissue) tissue) pos WT #seq #mut % mut mut #seq #mut % mut mut#seq #mut % mut mut #seq #mut % mut mut 55 L 29 0 0 50 5 10 5F 13 3 233F 37 2 5 2F 60 K 29 0 0 50 4 8 2M 13 1 8 1N 37 3 8 2M 1E 1E 1N 61 S 290 0 50 1 2 1L 13 0 0 37 1 3 1L 66 D 29 0 0 50 5 10 4H 13 0 0 37 5 14 4H1N 1N 69 E 29 0 0 50 1 2 1D 13 0 0 37 1 3 1D 74 N 29 0 0 50 4 8 4S 13 00 37 4 11 4S 75 K 29 0 0 50 3 6 3R 13 0 0 37 3 8 3R 113 I 41 3 7 3L 5018 36 18L 13 5 38 5L 37 13 35 13L 117 T 41 1 2 1S 50 8 16 8S 13 0 0 37 822 8S 123 S 41 0 0 50 4 8 4C 13 2 15 2C 37 2 5 2C 128 T 41 2 5 2A 50 612 6A 13 0 0 37 6 16 6A 134 A 41 20 49 20G 50 47 94 47G 13 10 77 10G 3737 100 37G 158 V 29 0 0 50 4 8 4L 13 0 0 37 4 11 4L 164 T 29 5 17 5K 5046 92 46K 13 10 77 10K 37 36 97 36K 223 V 42 0 0 52 1 2 1A 14 0 0 38 1 31A 265 N 42 0 0 52 4 8 3D 14 4 29 3D 38 0 0 1T 1T 267 S 42 0 0 52 4 8 3F14 1 7 1F 38 3 8 2F 1L 1L 269 S 42 0 0 52 12 23 9F 14 1 7 1F 38 11 29 8F3Y 3Y 271 Q 42 0 0 52 1 2 1H 14 0 0 38 1 3 1H 321 I 42 20 48 20V 32 2888 28V 5 1 20 1V 27 27 100 27V 332 Q 42 20 48 20E 32 29 91 29E 5 2 40 2E27 27 100 27E 345 R 42 0 0 32 8 25 8K 5 0 0 27 8 30 8K

TABLE 1B Panel of Group I JCV-VP1 variants PML patients PML PML Healthy(brain + patients patients JCV individ- peripheral (peripheral (brainVP1 uals tissue) tissue) tissue) pos WT #seq #mut % mut mut #seq #mut %mut mut #seq #mut % mut mut #seq #mut % mut mut 55 L 29 0 0 50 5 10 5F13 3 23 3F 37 2 5 2F 60 K 29 0 0 50 4 8 2M 13 1 8 1N 37 3 8 2M 1E 1E 1N61 S 29 0 0 50 1 2 1L 13 0 0 37 1 3 1L 66 D 29 0 0 50 5 10 4H 13 0 0 375 14 4H 1N 1N 69 E 29 0 0 50 1 2 1D 13 0 0 37 1 3 1D 74 N 29 0 0 50 4 84S 13 0 0 37 4 11 4S 75 K 29 0 0 50 3 6 3R 13 0 0 37 3 8 3R

TABLE 1C Panel of Group II JCV-VP1 variants PML patients PML PML Healthy(brain + patients patients JCV individ- peripheral (peripheral (brainVP1 uals tissue) tissue) tissue) pos WT #seq #mut % mut mut #seq #mut %mut mut #seq #mut % mut mut #seq #mut % mut mut 265 N 42 0 0 52 4 8 3D14 4 29 3D 38 0 0 1T 1T 267 S 42 0 0 52 4 8 3F 14 1 7 1F 38 3 8 2F 1L 1L269 S 42 0 0 52 12 23 9F 14 1 7 1F 38 11 29 8F 3Y 3Y 271 Q 42 0 0 52 1 21H 14 0 0 38 1 3 1H

TABLE 1D Panel of Group III JCV-VP1 variants PML patients PML PMLHealthy (brain + patients patients JCV individ- peripheral (peripheral(brain VP1 uals tissue) tissue) tissue) pos WT #seq #mut % mut mut #seq#mut % mut mut #seq #mut % mut mut #seq #mut % mut mut 113 I 41 3 7 3L50 18 36 18L 13 5 38 5L 37 13 35 13L 123 S 41 0 0 50 4 8 4C 13 2 15 2C37 2 5 2C 158 V 29 0 0 50 4 8 4L 13 0 0 37 4 11 4L 164 T 29 5 17 5K 5046 92 46K 13 10 77 10K 37 36 97 36K 345 R 42 0 0 32 8 25 8K 5 0 0 27 830 8K

TABLE 1E Panel of Groups I-III JCV-VP1 variants PML patients PML PMLHealthy (brain + patients patients JCV individ- peripheral (peripheral(brain VP1 uals tissue) tissue) tissue) pos WT #seq #mut % mut mut #seq#mut % mut mut #seq #mut % mut mut #seq #mut % mut mut 55 L 29 0 0 50 510 5F 13 3 23 3F 37 2 5 2F 60 K 29 0 0 50 4 8 2M 13 1 8 1N 37 3 8 2M 1E1E 1N 61 S 29 0 0 50 1 2 1L 13 0 0 37 1 3 1L 66 D 29 0 0 50 5 10 4H 13 00 37 5 14 4H 1N 1N 69 E 29 0 0 50 1 2 1D 13 0 0 37 1 3 1D 74 N 29 0 0 504 8 4S 13 0 0 37 4 11 4S 75 K 29 0 0 50 3 6 3R 13 0 0 37 3 8 3R 113 I 413 7 3L 50 18 36 18L 13 5 38 5L 37 13 35 13L 123 S 41 0 0 50 4 8 4C 13 215 2C 37 2 5 2C 158 V 29 0 0 50 4 8 4L 13 0 0 37 4 11 4L 164 T 29 5 175K 50 46 92 46K 13 10 77 10K 37 36 97 36K 265 N 42 0 0 52 4 8 3D 14 4 293D 38 0 0 1T 1T 267 S 42 0 0 52 4 8 3F 14 1 7 1F 38 3 8 2F 1L 1L 269 S42 0 0 52 12 23 9F 14 1 7 1F 38 11 29 8F 3Y 3Y 271 Q 42 0 0 52 1 2 1H 140 0 38 1 3 1H 345 R 42 0 0 32 8 25 8K 5 0 0 27 8 30 8K

TABLE 1F Panel of JCV-VP1 variants found in most PML brain samples PMLpatients PML PML Healthy (brain + patients patients JCV individ-peripheral (peripheral (brain VP1 uals tissue) tissue) tissue) pos WT#seq #mut % mut mut #seq #mut % mut mut #seq #mut % mut mut #seq #mut %mut mut 134 A 41 20 49 20G 50 47 94 47G 13 10 77 10G 37 37 100 37G 164 T29 5 17 5K 50 46 92 46K 13 10 77 10K 37 36 97 36K 321 I 42 20 48 20V 3228 88 28V 5 1 20 1V 27 27 100 27V 332 Q 42 20 48 20E 32 29 91 29E 5 2 402E 27 27 100 27E

TABLE 1G Panel of JCV-VP1 risk variants no found in samples from healthyindividuals PML patients PML PML Healthy (brain + patients patients JCVindivid- peripheral (peripheral (brain VP1 uals tissue) tissue) tissue)pos WT #seq #mut % mut mut #seq #mut % mut mut #seq #mut % mut mut #seq#mut % mut mut 55 L 29 0 0 50 5 10 5F 13 3 23 3F 37 2 5 2F 60 K 29 0 050 4 8 2M 13 1 8 1N 37 3 8 2M 1E 1E 1N 61 S 29 0 0 50 1 2 1L 13 0 0 37 13 1L 66 D 29 0 0 50 5 10 4H 13 0 0 37 5 14 4H 1N 1N 69 E 29 0 0 50 1 21D 13 0 0 37 1 3 1D 74 N 29 0 0 50 4 8 4S 13 0 0 37 4 11 4S 75 K 29 0 050 3 6 3R 13 0 0 37 3 8 3R 123 S 41 0 0 50 4 8 4C 13 2 15 2C 37 2 5 2C158 V 29 0 0 50 4 8 4L 13 0 0 37 4 11 4L 223 V 42 0 0 52 1 2 1A 14 0 038 1 3 1A 265 N 42 0 0 52 4 8 3D 14 4 29 3D 38 0 0 1T 1T 267 S 42 0 0 524 8 3F 14 1 7 1F 38 3 8 2F 1L 1L 269 S 42 0 0 52 12 23 9F 14 1 7 1F 3811 29 8F 3Y 3Y 271 Q 42 0 0 52 1 2 1H 14 0 0 38 1 3 1H 345 R 42 0 0 32 825 8K 5 0 0 27 8 30 8K

TABLE 1H Panel of JCV-VP1 variants found in healthy individuals PMLpatients PML PML Healthy (brain + patients patients JCV individ-peripheral (peripheral (brain VP1 uals tissue) tissue) tissue) pos WT#seq #mut % mut mut #seq #mut % mut mut #seq #mut % mut mut #seq #mut %mut mut 115 V 41 1 2 1E 50 0 0 13 0 0 37 0 0 277 R 42 1 2 1K 52 0 0 14 00 38 0 0

TABLE 2 A: Healthy Subjects SEQUENCE #MUT #REGION LENGTH VARIANTSAAQ88264_gi|37050849|gb|AAQ 0 0 347 CONSENSUSAAB60589_gi|1161330|gb|AAB6 2 0 27 128 A 134 GAAC54623_gi|1161332|gb|AAC5 0 0 27 AAG48112_gi|12056896|gb|AAG 2 0 142321 V 332 E AAG48114_gi|12056899|gb|AAG 2 0 142 321 V 332 EAAG48119_gi|12056907|gb|AAG 2 0 142 321 V 332 EAAG48121_gi|12056910|gb|AAG 2 0 142 321 V 332 EAAG48123_gi|12056913|gb|AAG 2 0 142 321 V 332 EAAG48125_gi|12056916|gb|AAG 2 0 142 321 V 332 EAAG48127_gi|12056919|gb|AAG 2 0 142 321 V 332 EAAG48544_gi|12082440|gb|AAG 1 0 56 134 G AAG48545_gi|12082442|gb|AAG 1 056 134 G AAG48546_gi|12082444|gb|AAG 1 0 56 134 GAAG48547_gi|12082446|gb|AAG 1 0 56 134 G AAG48548_gi|12082448|gb|AAG 1 056 134 G AAG48549_gi|12082450|gb|AAG 2 1 56 113 L 134 GAAG48550_gi|12082452|gb|AAG 2 1 56 113 L 134 GAAG48551_gi|12082454|gb|AAG 2 1 56 113 L 134 GAAL12481_gi|16118426|gb|AAL 2 0 142 321 V 332 EAAL12483_gi|16118429|gb|AAL 2 0 142 321 V 332 EAAR06661_gi|37993002|gb|AAR 0 0 347 AAR12957_gi|38156673|gb|AAR 1 0 347134 G AAR13077_gi|38176429|gb|AAR 1 0 347 134 GAAR13659_gi|38195910|gb|AAR 0 0 347 AAR32743_gi|39939397|gb|AAR 0 0 347AAR89187_gi|40737215|gb|AAR 0 0 347 AAR89193_gi|40737222|gb|AAR 0 0 347AAR89199_gi|40737229|gb|AAR 0 0 347 AAR89205_gi|40737236|gb|AAR 0 0 347AAR89211_gi|40737243|gb|AAR 1 0 347 277 K AAR89217_gi|40737250|gb|AAR 00 347 AAR89223_gi|40737257|gb|AAR 0 0 347 AAR89229_gi|40737264|gb|AAR 00 347 AAR89235_gi|40737271|gb|AAR 0 0 347 AAR89241_gi|40737278|gb|AAR 00 347 AAR89247_gi|40737285|gb|AAR 0 0 347 AAR89253_gi|40737292|gb|AAR 00 347 AAR89259_gi|40737299|gb|AAR 1 0 347 134 GAAR89265_gi|40737543|gb|AAR 0 0 347 AAR89271_gi|40737550|gb|AAR 1 0 347115 E AAR89277_gi|40737557|gb|AAR 0 0 347 AAR89283_gi|40737564|gb|AAR 00 347 ABA60111_gi|76885940|gb|ABA 0 0 58 BAA01963_gi|425204|dbj|BAA0 4 1347 134 G 164 K 321 V 332 E BAA01964_gi|425205|dbj|BAA0 4 1 347 134 G164 K 321 V 332 E BAA07834_gi|1772312|dbj|BAA 2 0 134 321 V 332 EBAA07836_gi|1785835|dbj|BAA 2 0 134 321 V 332 EBAA07838_gi|1785837|dbj|BAA 2 0 134 321 V 332 EBAA07840_gi|1772321|dbj|BAA 2 0 134 321 V 332 EBAB11698_gi|9796401|dbj|BAB 4 1 347 134 G 164 K 321 V 332 EBAB11704_gi|9796408|dbj|BAB 4 1 347 134 G 164 K 321 V 332 EBAB11716_gi|9796422|dbj|BAB 3 0 347 134 G 321 V 332 EBAB11722_gi|9796429|dbj|BAB 3 0 347 134 G 321 V 332 E Average Values 1.20.1 234.7 B: PML Samples Sequence #mut #region Length VariantsAAB62680_gi|2246607|gb|AA 5 1 347 128 A 134 G 164 K 321 V 332 EAAT09819_gi|47078338|gb|A 5 3 347 113 L 134 G 164 K 265 T 332 EAAT09825_gi|47078345|gb|A 1 1 347 267 F AAT09831_gi|47078352|gb|A 1 1347 55 F AAT09837_gi|47078359|gb|A 1 1 347 60 NBAE02848_gi|68445641|dbj| 4 3 246 113 L 134 G 164 K 265 DBAE02849_gi|68445643|dbj| 3 2 246 55 F 134 G 164 KBAE02850_gi|68445645|dbj| 4 3 246 113 L 134 G 164 K 265 DBAE02851_gi|68445647|dbj| 4 3 246 113 L 123 C 134 G 164 KBAE02852_gi|68445649|dbj| 3 2 246 134 G 164 K 269 FBAE02853_gi|68445651|dbj| 4 3 246 113 L 123 C 134 G 164 KBAE02854_gi|68445653|dbj| 3 2 246 55 F 134 G 164 KBAE02855_gi|68445655|dbj| 2 1 246 134 G 164 K BAE02856_gi|68445657|dbj|3 2 246 134 G 164 K 265 D AAB60586_gi|1161322|gb|AAB 2 0 134 321 V 332 EPartial AAB60584_gi|1161319|gb|AAB 2 0 134 321 V 332 E PartialAAB62687_gi|2246615|gb|AAB 6 2 347 55 F 128 A 134 G 164 K 321 V 332 EAAB94036_gi|2735983|gb|AAB 4 1 347 55 F 134 G 321 V 332 EBAA01965_gi|425206|dbj|BAA 6 2 347 128 A 134 G 164 K 269 F 321 V 332 EBAA01966_gi|425207|dbj|BAA 9 5 347 66 H 75 R 117 S 134 G 158 L 164 K 321V 332 E 345 K BAA01967_gi|425208|dbj|BAA 8 4 347 117 S 134 G 158 L 164 K267 L 321 V 332 E 345 K BAA01968_gi|425209|dbj|BAA 8 3 347 74 S 117S 128A 134 G 164 K 321 V 332 E 345 K BAA01969_gi|425210|dbj|BAA 6 3 347 113 L134 G 164 K 269 F 321 V 332 E BAA01970_gi|425211|dbj|BAA 5 2 347 113 L134 G 164 K 321 V 332 E BAA05636_gi|538231|dbj|BAA 5 2 347 113 L 134 G164 K 321 V 332 E BAA05637_gi|538234|dbj|BAA 6 3 347 113 L 134 G 164 K269 F 321 V 332 E BAA05638_gi|538238|dbj|BAA 5 2 347 134 G 164 K 269 Y321 V 332 E BAB11728_gi|9796436|dbj|BA 6 3 347 113 L 134 G 164 K 269 F321 V 332 E BAB11734_gi|9796443|dbj|BA 5 2 347 134 G 164 K 269 Y 321 V332 E BAE00111_gi|67968154|dbj|B 8 3 347 74 S 117 S 128 A 134 G 164 K321 V 332 E 345 K BAE00117_gi|67968161|dbj|B 9 4 347 74 S 117 S 128 A134 G 164 K 269 F 321 V 332 E 337 K BAE00123_gi|67968168|dbj|B 9 4 34774 S 117 S 128 A 134 G 164 K 269 F 321 V 332 E 337 KBAE00129_gi|67968175|dbj|B 4 1 347 134 G 164 K 321 V 332 EBAE00135_gi|67968182|dbj|B 5 2 347 61 L 134 G 164 K 321 V 332 EBAE00141_gi|67968189|dbj|B 4 1 347 134 G 164 K 321 V 332 EBAE00147_gi|67968204|dbj|B 5 2 347 113 L 134 G 164 K 321 V 332 EBAE00153_gi|67968211|dbj|B 6 3 347 113 L 134 G 164 K 269 Y 321 V 332 EBAE00159_gi|67968218|dbj|B 6 3 347 60 M 113 L 134 G 164 K 321 V 332 EBAE00165_gi|67968225|dbj|B 6 3 347 113 L 123 C 134 G 164 K 321 V 332 EBAE00171_gi|67968232|dbj|B 6 3 347 105 L 123 C 134 G 164 K 321 V 332 EBAE02837_gi|68445619|dbj|B 2 1 246 134 G 164 KBAE02838_gi|68445621|dbj|B 3 2 246 66 H 134 G 164 KBAE02839_gi|68445623|dbj|B 3 2 246 66 H 134 G 164 KBAE02840_gi|68445625|dbj|B 4 3 246 60 E 66 H 134 G 164 KBAE02841_gi|68445627|dbj|B 4 3 246 113 L 134 G 164 K 267 FBAE02842_gi|68445629|dbj|B 4 3 246 60 M 134 G 164 K 270 HBAE02843_gi|68445631|dbj|B 3 2 246 66 H 134 G 164 KBAE02844_gi|68445633|dbj|B 4 3 246 113 L 134 G 164 K 269 FBAE02845_gi|68445635|dbj|B 4 3 246 113 L 134 G 164 K 267 FBAE02846_gi|68445637|dbj|B 3 1 246 134 G 164 K 223 ABAE02847_gi|68445639|dbj|B 4 3 246 69 D 134 G 164 K 269 FP03089_gi|116626|sp|P03089 8 4 347 75 R 117 S 134 G 158 L 164 K 321 V332 E 345 K BAB11710_gi|9796415|dbj|BAB 8 4 347 75 R 117 S 134 G 158 L164 K 321 V 332 E 345 K MAD-1 Average values 4.7 2.4 300.8

Diagnostics

Accordingly, methods of the invention are useful in one aspect fordetermining if a subject is at risk for, or has, PML based on the panelsand groups of variant/variants of the invention. Assaying a biologicalsample from a subject for an indicium of exposure to a variant JCV willallow for the determination if a person is at risk for, or has, PML, oris infected by a JCV variant associated with PML. The determination ofwhether a subject has been exposed to, or is infected with, a JCVvariant having a sequence variation at one or more of the VP1 positionsdescribed herein allows for the assessment of whether a subject is atrisk for PML (variants at VP1 positions of a JCV variant associated withPML are also referred to as variants of the invention). In one aspect,the invention provides correlations between the mutational status atpredetermined positions of a JCV variant and risk for PML. In someembodiments the variants are located on the JCV-VP1 protein.

As used herein, “diagnosing” and “identifying” PML means the recognitionof whether a person is at risk for PML, or has PML. The diagnosis ofbeing at risk for PML is not limited to the determination of variants atpositions indicative of exposure to a JCV variant and may be combinedwith diagnosis methods routine in the art. These diagnostic assaysinclude but are not limited to histopathology, immunohistochemistry,flow cytometry, cytology, patho-physiological assays, including MRI andtomography, neurological assays biochemical assays. Detection of JCV DNAby PCR in CSF is the most widely accepted diagnostic test of PML. It has99% specificity and 70% selectivity. In the absence of a positive JCVPCR result for CSF, a brain biopsy could be performed. JCV DNA detectionin brain tissue can be used as a positive diagnosis of PML. Biochemicalassays include but are not limited to variant analysis other than at thepredetermined positions, viral genome analysis, ELISA analysis ofspecific proteins, platelet count etc. Those of ordinary skill in theart will be aware of numerous diagnostic protocols and parameters thatare routinely utilized in the art.

Diagnosis also covers a determination of the amount of JCV variant(viral load) and the ratio of JCV variant versus JCV wild type in asample and/or subject.

Methods and/or kits of the invention can be used to screen subjects forhaving PML and being at risk for PML as indicated by the presence ofvariants at predetermined positions indicative of exposure to a JCVvariant (the variants of the invention), in which the presence of one ormore variants of the invention is associated with being at risk for PML.Methods of the invention may be used to diagnose the risk for PML byassessing the variants of the invention in a sample from a subject thathas been exposed or is suspected of having been exposed to a JCV variantassociated with PML.

The invention, in some aspects, includes various assays to determinewhether a subject has been exposed to a JCV variant having one or morevariants at specific positions in the VP1 protein. Methods and assays ofthe invention may be used to monitor changes in the status of variantsof the invention in a sample and or a subject over time. In addition,methods of the invention may be used to monitor the amount of JCVvariant present in subject over time, by obtaining multiple sample fromthe subject at different time points. Thus, methods of the invention maybe used to examine changes in variants of the invention and/or amount ofJCV variant in a subject or sample over time. This allows monitoring ofmutational status in a subject who is suspected to be at risk ofdeveloping PML and also enables quantitative monitoring in a subject whois known to have been exposed to a JCV variant.

Detection of JCV-VP1 Variant Nucleic Acids, Polypeptides and Antibodies

In one aspect the invention provides methods for the detection ofJCV-VP1 variant polypeptides and/or antibodies that can specificallybind the JCV variant polypeptides in a biological sample (also referredto as polypeptides and antibodies of the invention, respectively).Methods of detecting polypeptides and antibodies are well known in theart and the invention is not limited to any specific detection method.In addition, the polypeptides and antibodies may be detected indirectly,through the detection of nucleic acids encoding the polypeptides orantibodies of the invention. Methods for the detection of nucleic acidsare well known in the art and non-limiting examples are PCR, sequencing,hybridization analysis, probe analysis and microarray analysis, whichare all embraced by the current invention. Non-limiting examples ofmethods for the detection of polypeptides and/or antibodies includepeptide sequencing, mass spectrometry and immunosorbent assays (e.g.,binding of the polypeptide to an antibody) and protein arrays.

Enzyme-linked immunosorbent assays (ELISAs) are a well-known assays usedfor the detection of various antigens including polypeptides. Theinvention embraces any ELISA that can detect the presence of apolypeptides of the invention and any ELISA that can detect the presenceof antibodies of the invention in a sample.

In one embodiment a first step of an ELISA comprises providing a primaryantibody specific for a JCV-VP1 polypeptide bound to a multiwell plate.The invention is not limited to multiwell plates and the antibodies maybe immobilized onto any surface including beads and glass slides. Theinvention is also not limited to antibodies and any peptide bindingmoiety, including aptamers, antibody fragments and small molecules areembraced by the invention. The invention embraces both the qualitativeand quantitative determination of the presence of polypeptides of theinvention. In one embodiment, the wells can be blocked using a buffercontaining a high concentration of irrelevant protein, such as bovineserum albumin or casein. The blocking step ensures that any uncoatedareas of the surface will be occupied with non-reactive protein, ifneeded. Excess blocking agent is then removed by one or more washes. Insome embodiments the ELISA is a multiplex assay and each well of amultiwell plate comprises an antibody specific for one specific JCV-VP1polypeptide variant. Once the surface is blocked, a sample containing aJCV-VP1 polypeptide, or a sample to be tested for presence of a JCV-VP1polypeptide, can be contacted with the primary antibody and is allowedto incubate under conditions and for an amount of time suitable topermit specific binding of the JCV-VP1 polypeptide to the primaryantibody. Such conditions and amount of time can be, for example, roomtemperature for 3-4 hours, or 4° C. for 10-16 hours. Excess sample isthen removed by one or more washes. As a next step, a secondaryantibody, also specific for the JCV-VP1 polypeptide, is contacted to theprimary antibody-polypeptide complex and is allowed to incubate underconditions and for an amount of time suitable to permit specific bindingof the JCV-VP1 polypeptide by the secondary antibody. Such conditionsand amount of time can be, for example, antibody at 1-10 microgram/ml,room temperature for 1-4 hours, and 4° C. for 10-16 hours. In someembodiments, the secondary antibody is connected to a fluorescent tag orto a metabolizing enzyme, allowing for the detection of bound JCV-VP1polypeptide. Alternatively, bound JCV-VP1 polypeptide can be determinedby contacting the secondary antibody with a labeled tertiary antibody.The above-described ELISA is referred to as a sandwich ELISA as theJCV-VP1 polypeptide is sandwiched between two antibodies (the primaryantibody and the secondary antibody).

In some embodiments, the presence of antibodies specific for JCV-VP1polypeptides is determined. The invention embraces both the qualitativeand quantitative determination of presence of antibodies of theinvention. Determination of the presence of antibodies can allow for thedetermination of both current and past infection. In most instances, thehuman body will develop an antibody against a foreign polypeptide, suchas a JCV proteins. Even if the virus (e.g., viral nucleic acid) itselfis no longer detected in the body, antibodies against the virus maystill be present, thereby being an indicator of exposure to the virus.In one embodiment of a method for detecting antibodies specific forvariant JCV-VP1 polypeptides, one or more solid surfaces (e.g., amulti-well plate, bead or slide) are provided wherein each surface hasan immobilized unique JCV-VP1 polypeptide variant.

In some embodiments the JCV-VP1 polypeptide variants are immobilized asJCV particles (e.g., recombinant VP1 proteins that contain one or morevariants and that are self-assembled to form particles that areimmobilized, for example, on an assay surface). These particles maycontain one or more of the variants described herein (see, e.g., Tables1A-1H, Table 17 and/or Table 18, and/or one or more other variantsdescribed herein).

Each polypeptide variant comprises one or more of the variants of Tables1A-1H, Table 17 and/or Table 18 and/or one or more other variantsdescribed herein. Multiple peptides may cover the same one or morevariants. In one embodiment combinations of polypeptides variants areattached to the solid surface. In some embodiments the solid surface amulti-well plate is provided wherein each well comprises one or morevariants. In some embodiments a multi-well plate comprises wells withpeptides covering all variants described herein that are indicative ofrisk for PML. A sample comprising or suspected of comprising one or moreantibodies that can bind to a JCV-VP1 Polypeptide variant describedherein is contacted with the immobilized peptides under conditionssuitable for the antibodies to bind to the immobilized JCV-VP1polypeptide. The presence of the bound antibodies is subsequentlydetected through binding of a secondary antibody.

The ELISAs of the invention are not limited to the above describedembodiments, but also embraces competitive ELISAs and any form of ELISAthat allows for the detection of the polypeptides and antibodies of theinvention.

Onset, Progression, Regression

Methods and/or kits of the invention can be used to obtain usefulprognostic information by providing an early indicator of PML onset,progression, and/or regression. The invention includes methods tomonitor the onset, progression, or regression of PML and/or JCV variantinfection in a subject by, for example, obtaining samples at sequentialtimes from a subject and assaying such samples for exposure to JCVvariants of the invention. A subject may be suspected of having PML ormay be believed not to have PML and in the latter case, the sample mayserve as a normal baseline level for comparison with subsequent samples.

Onset of a condition is the initiation of the changes associated withthe condition in a subject. Such changes may be evidenced byphysiological symptoms. However, a patient may be clinicallyasymptomatic. For example, the onset of PML and/or JCV infection may befollowed by a period during which there may be PML-associated pathogenicchanges in the subject, even though clinical symptoms may not be evidentat that time. The progression of PML follows onset and is theadvancement of the pathogenic (e.g., physiological) elements of thecondition, which may or may not be marked by an increase in clinicalsymptoms. In contrast, the regression of PML and/or JCV infection mayinclude a decrease in physiological characteristics of the condition,perhaps with a parallel reduction in symptoms, and may result from atreatment or may be a natural reversal in the condition. Onset of PMLand/or JCV infection may be indicated by a change in the variants at theinterrogated positions in samples obtained from the subject. Forexample, if the number of variants at the interrogated positions islower in a first sample from a subject, than in a second or subsequentsample from the subject, it may indicate the onset or progression of PMLand/or JCV infection. In some embodiments the presence of only onevariant may be indicative of the onset or progression of PML. In someembodiments the variant is at position 164. In a particular embodiment,the emergence of a variant amino acid (e.g., Lys) at position 164 isindicative of onset or progression of PML. In some embodiments onset ofPML and/or JCV infection is indicated by a change in viral load of JCVvariant and/or ratio of JCV variant to JCV wild type. In someembodiments an increase in viral load is accompanied by an increase inthe number of variants.

Progression and regression of PML and/or JCV infection may be generallyindicated by the increase or decrease in the number of variants atinterrogated positions in a subject's samples over time. For example, ifthe number of variants is low in a first sample from a subject andincreased levels of variants are determined to be present in a second orsubsequent sample from the subject, it may indicate the progression ofPML and/or JCV infection, respectively. It should be appreciated thatboth an increase and a decrease in the number of variants, may beindicative of progression or regression of PML and/or JCV infection,respectively. For instance, the number of variants at certain positionsmay increase, while the number of variants at other positions maydecrease, with both changes being indicative of PML and/or JCVinfection.

Progression of PML and/or JCV infection may also be indicated by theincrease in viral load of a JCV variant or an increase in the ratio ofJCV variant to JCV wild type, while regression may be indicated by adecrease in viral load of a JCV variant or a decrease in the ratio ofJCV variant to JCV wild type.

Onset, progression and regression can be determined by assaying multiplesamples that are obtained from a subject at different time points. Forinstance, even if a first group of samples taken at different timepoints does not show onset, progression or regression of PML anadditional sample may be obtained and assayed for the variants of theinvention and compared to earlier samples to determine whether onset,progression or regression are occurring. In some embodiments additionalsamples are assayed when the administration regimes of one or moreimmunosuppressants is modified.

Assays for PML Treatment

Methods of the invention may also be used to assess the efficacy of atherapeutic treatment of PML by interrogation for exposure to a variantof JC virus in a subject at various time points. For example, the numberof variants indicative of exposure to a variant JC virus can be obtainedprior to the start of a therapeutic regimen (either prophylactic or as atreatment of cancer or a precancerous condition), during the treatmentregimen, and/or after a treatment regimen, thus providing information onthe effectiveness of the regimen in the subject. In addition, theabundance of JCV variants indicative of PML risk can also be monitoredover time. In some embodiments, the amount of JCV variant (viral load)or ratio of JCV variant to wild type JCV may provides information on theeffectiveness of the regimen in the subject. Methods of the inventionmay be used to compare the mutated positions in two or more samplesobtained from a subject at different times. In some embodiments, asample is obtained from a subject, the subject is administered atreatment for PML and a subsequent sample is obtained from the subject.A comparison of a subject's variants of the invention or viral load ofthe JC variant or ratio of JC variant to JCV wild type is determined insamples obtained at different times and/or on different days, therebyproviding a measure of the status of the subject's PML, which can beused to determine the effectiveness of any treatment for PML and/or JCVinfection in a subject.

As used herein, PML treatment encompasses both prophylactic andtherapeutic treatment, and it embraces both the prevention and treatmentof PML. A subject can receive PML treatment because the subject has beendetermined to be at risk for PML or alternatively, the subject may havePML. Thus, a treatment may reduce or eliminate PML and/or JCV infectionaltogether or prevent it from becoming worse. “Evaluation of treatment”as used herein, means the comparison of a subject's levels of variantsindicative for exposure to a JCV variant or the viral load of the JCVvariant in samples obtained from the subject at different sample times,for example, at least one day apart. In some embodiments, the time toobtain the second sample from the subject is at least 5, 10, 20, 30, 40,50, minutes after obtaining the first sample from the subject. Incertain embodiments, the time to obtain the second sample from thesubject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, 96, 120 or more hoursafter obtaining the first sample from the subject. In some embodiments,the time to obtain the second sample from the subject is at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28,35, 42, 49, 56, 60, 90, 120, 150, 180, 360 or more days after obtainingthe first sample from the subject.

Treatment of PML

In one embodiment, a therapeutic agent for the treatment of PML is ananti-infective agent. A therapeutic agent may be an anti-infective agentthat treats a microbial infection, e.g., a therapeutic agent that iseffective to treat an infection of the CNS. In some embodiments, ananti-infective agent may be an siRNA. However, other suitableanti-infective agents may be used. Anti-infective agents may beantimicrobial agents such as antiviral agents. Antiviral agents may becombined with additional anti-infective agents such as antifungal agentsor antibacterial agents. Therapeutic agents of the invention may benaturally occurring or synthetic molecules or compounds. A therapeuticagent useful in methods and compositions of the invention may be apolypeptide, chemical, small molecule, lipid, nucleic acid, or othercompound.

Examples of nucleic acids that can be used as therapeutic agents (e.g.,anti-infective agents) in methods and compositions of the invention aresmall interfering RNA molecules (siRNA), antisense DNA, antisense RNA,or aptamers that can be used to prevent and/or treat a CNS disease ordisorder, including PML. A “small interfering RNA” or “siRNA,” as usedherein, refers to a RNA molecule that can be derived from the successivecleavage of a double-stranded RNA (dsRNA) within a cell to produce anRNA molecule. An effective siRNA generally has a length of between 15and 30 nucleotides, and more often between 20 and 25 nucleotides. siRNAsfunction to direct the destruction of corresponding mRNA targets duringRNA interference in animals. Methods of the invention, in part, includethe administration of siRNA molecules to treat a CNS disease or disorder(e.g., a CNS infection) associated with a variant JC virus. In someaspects, methods of the invention may include administering to a subjecta therapeutic composition that includes an siRNA and/or a precursorsiRNA as a treatment of a CNS disease or condition. A precursor siRNAmolecule is a double-stranded RNA molecule that after administration canbe reduced in size, for example by the enzyme Dicer, to form theintended siRNA, this becoming functional for RNAi treatment of a CNSdisease or condition. Thus, methods of the invention may includeadministration of an siRNA or a precursor siRNA molecule to treat a CNSdisease or condition.

Selecting Treatment

In some embodiments, methods of the invention may be used to help selecta treatment for a subject with PML or at risk for PML. Selection of atreatment for PML may be based upon the determination of the variantsindicative of exposure to a JCV variant. Selection of treatment may alsobe based on the amount of JCV variant in a sample (viral load) or on theratio of variant JCV to wildtype JCV. Methods of selecting a treatmentmay be useful to assess and/or adjust treatment of subjects alreadyreceiving a drug or therapy for PML. Based on the variants found thatare indicative of exposure to a JCV variant, it may be appropriate toalter a therapeutic regimen for a subject. For example, detection of achange in one or more of the positions indicative of exposure to a JCVvariant in a subject who has received or is receiving PML or JCVtreatment may indicate that the treatment regimen should be adjusted(e.g., the dose or frequency of dosing, increased, new treatmentinitiated, etc.). In some embodiments, the change in viral load of a JCVvariant or ratio of variant JCV to wildtype JCV may indicate that thetreatment regimen should be adjusted. In some embodiments, a subject maybe free of any present treatment for PML and/or JCV infection andmonitoring of variants at positions indicative of exposure to JCVvariant and/or viral load of the JCV variant and/or the ratio of JCVvariant to JCV wildtype may identify the subject as a candidate for atreatment for PML and or JCV infection. Thus, subjects may be selectedand treated with elevated levels of the same drugs or with differenttherapies as a result of assays for indicia of exposure to a JCV variantand/or a viral load of the JCV variant and/or a ratio of JCV variant toJCV wild type.

According to the present invention, some subjects may be free ofsymptoms otherwise calling for treatment with a particular therapy, andthe detection of indicia of exposure to a JCV variant and/or a viralload of a JCV variant and/or a ratio of JCV variant to JCV wild type mayidentify the subject as needing treatment. This means that absent theuse of the methods of the invention to identify indicia of exposure to aJCV variant and/or a viral load of a JCV variant and/or a ratio of JCVvariant to JCV wild type, the subject would not according to conventionas of the date of the filing of the present application have symptomscalling for treatment with a particular PML and/or JCV infectiontherapy.

In one aspect, the invention provides methods for the decision ontreatment regimens for PML. A clinician can make decisions on treatmentoptions at least in part based on the diagnostic assays of theinvention.

Treatment Relating to Immunosuppressants

Methods of selecting treatment may be useful for persons undergoingtreatment not directed to PML or JCV infection, but directed to adifferent condition. In one embodiment, the treatment is a treatmentcomprising immunosuppressants. In some embodiments, a person suspectedof being at risk for developing PML is a person undergoing treatmentwith immunosuppressants.

In some embodiments, detection of a change in one or more indicia ofexposure to a JCV variant in a subject who has received or is receivingtreatment not directed to PML, may indicate that the treatment regimenshould be adjusted. In some embodiments, detection of a change in one ormore indicia of exposure to a JCV variant in a subject who has receivedor is receiving treatment with immunosuppressants may indicate that thetreatment regimen should be adjusted. In some embodiments, detection ofa change in viral load of a JCV variant or ratio of JCV variant to wildtype in a subject who has received or is receiving treatment withimmunosuppressants may indicate that the treatment regimen should beadjusted. In some embodiments, detection of a sequence change in one ormore predetermined VP1 positions of a JCV in a subject who has receivedor is receiving treatment with immunosuppressants may indicate that thetreatment regimen should be terminated or interrupted. In someembodiments, the immunosuppressant is natalizumab. In some embodiments,detection of an increase in the viral load of a JCV variant and/or theratio of JCV variant to JCV wild type in a subject who has received oris receiving treatment with immunosuppressants may indicate that thetreatment regimen should be terminated or interrupted.

In one aspect, the invention provides methods for the decision ontreatment regimens. A clinician can make decisions on treatment optionsat least in part based on the diagnostic assays of the invention. Insome embodiments the methods of the invention provide a diagnostic assayto decide on a suitable treatment with immunosuppressive agents. In someembodiments, a clinician may decide to terminate or interrupt treatmentwith immunosuppressive agents based on the outcome of one or moreJCV-VP1 diagnostic assays of the invention. In some embodiments, themethods of the invention provide a diagnostic assay for the treatment ofa subject, wherein the subject is immuno-compromised. In someembodiments, a clinician may decide to terminate or interrupt treatmentof the immuno-compromised subject based on the outcome of a JCV-VP1diagnostic assays of the invention

Immuno-Compromised Subjects or Subjects Undergoing Treatment withImmuno-Suppressants

Assays of the invention may be particularly useful forimmuno-compromised subjects and/or subjects being treated with one ormore immuno-suppressants.

Subjects may receive treatment with one or more immunosuppressive agents(also called immuno-suppressants) directed to different diseases orconditions, including one or more of the following non-limitingexamples: cancer, organ or tissue transplant, inflammatory conditions ordiseases, multiple sclerosis (MS), arthritis, etc., or any combinationthereof.

Subjects may also be immuno-compromised. Non-limiting examples ofimmuno-compromised subjects are subject that are HIV positive or haveAIDS or lymphoma or any other condition resulting in a suppression ofthe immune response.

The term “immuno-suppressive agent” as used herein refers to substancesthat act to suppress or mask the immune system of a subject beingtreated herein. Immuno-suppressive agents may be substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,glucocorticoids such as cortisol or aldosterone, anti-inflammatoryagents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as corticosteroids or glucocorticosteroidsor glucocorticoid analogs, e.g., prednisone, methylprednisolone, anddexamethasone; dihydrofolate reductase inhibitors such as methotrexate(oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide;cytokine or cytokine receptor antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosisfactor-alpha antibodies (infliximab or adalimumab), anti-TNF-alphaimmunoahesin (etanercept), anti-tumor necrosis factor-beta antibodies,anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies;anti-CD20 antibodies (e.g., rituximab, for example available under thetrademark RITUXAN); anti-L3T4 antibodies; anti-VLA-4 antibodies (e.g.,natalizumab, for example available under the trademark TYSABR1);heterologous anti-lymphocyte globulin; pan-T antibodies, for exampleanti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3binding domain (WO 90/08187 published Jul. 26, 1990); streptokinase;TGF-beta; streptodornase; RNA or DNA from the host; FK506; RS-61443;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al., Science, 251:430432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.However, subjects receiving other immunosupressive agents may selectedfor diagnostic assays and/or treated as the invention is not limited inthis respect.

Screening for Candidate Therapeutic Agents

The invention also embraces methods for screening for candidatetherapeutic agents or strategies to prevent and/or treat PML and/or JCVinfection. Assessment of the efficacy of candidate therapeutic agentsand strategies may be done using assays of the invention in subjects(e.g., non-human animals) and in cells from culture. In someembodiments, a candidate therapeutic agent may be a compound or moleculethat can interact with a polypeptide comprising one or more JCV-VP1variants associated with PML. In some embodiments, a candidatetherapeutic agent may be a compound or molecule that can change thepattern and number of JCV-VP1 variants in a subject having a JCVinfection. In some embodiments, a candidate therapeutic is an agent thatcan reduce the amount of JCV variant (viral load) in a sample orsubject. In some embodiments, a candidate therapeutic is an agent thatcan reduce the ratio of variant JCV to wild type JCV. In someembodiments, a candidate therapeutic is an agent (e.g., a smallmolecule) that mimics the JCV receptor and can compete for JCV binding.In some embodiments, administration to the subject of an agent, orexposing a sample to the candidate agent or candidate treatment, willresult in a change in the pattern of JCV-VP1 variants associated with aJCV infection in a subject. In some embodiments, administering to thesubject of an agent, or exposing a sample to the candidate agent orcandidate treatment, will result in a change in the number of PMLassociated JCV-VP1 variants in a JCV infection. In some embodiments,administering to the subject of an agent, or exposing a sample to thecandidate agent or candidate treatment, will result in a decrease of JCVvariant viral load and/or ratio of JCV variant to JCV wild type.

In one embodiment, a sample comprising cells expressing a JCV variant isexposed to a candidate therapeutic agent or treatment. A samplecomprising cells exposed to the candidate, that do not express the JCVvariant will function as a control. The level of expression of the JCVvariant (viral load) is monitored upon administration of the candidatetherapeutic or treatment, and the change in expression of the JCVvariant is compared to samples that did not get treated with thecandidate therapeutic agent. If the level of viral load of the JCVvariant of the invention in the sample that has been exposed to theagent has changed compared with the control sample, the agent is acandidate therapeutic or candidate treatment. In some embodiments JCVvariants are expressed and secreted from cells and subjected tocontacting with a candidate agent. Any candidate agent that can bind theJCV variant is a candidate therapeutic agent. In some embodiments, thecandidate therapeutic agent binds to a VP1 polypeptide of the JCVvariant.

In some embodiments, the invention provides methods for identifyingcandidate therapeutic agents that suppress the number or level ofPML-associated VP1 variants in a JCV infection, and that suppress therisk for PML. In some embodiments, the candidate therapeutic agents aredirected to a disease or condition that is not PML or JCV infection. Insome embodiments, the candidate therapeutic agents areimmunosuppressants. In some embodiments, the candidate therapeuticagents are agents that are being prescribed in immunosuppressanttherapy.

Methods of screening for agents or treatments that modulate levels ofexpression of JCV variants are encompassed by the invention. Screeningmethods may include mixing the candidate agent with cells or tissues orin a subject or exposing cells or tissues or a subject to the candidatetreatment and using methods of assaying viral load of the JCV variantsto determine the level of expression before and after contact with thecandidate agent or treatment. A decrease in the amount of expression ofthe JCV variant compared to a control is indicative that the candidateagent or treatment is capable of treating PML and/or JCV infection in acell, tissue, and/or subject.

In some embodiments, an assay mixture for testing a candidate agentcomprises a candidate agent. A candidate agent may be an antibody, asmall organic compound, or a polypeptide, and accordingly can beselected from combinatorial antibody libraries, combinatorial proteinlibraries, or small organic molecule libraries. Typically, pluralitiesof reaction mixtures are run in parallel with different agentconcentrations to obtain a different response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, e.g., at zero concentration of agent or at aconcentration of agent below the limits of assay detection.

Any molecule or compound can be a candidate therapeutic. Non-limitingexamples of candidate therapeutics are small molecules, RNA includingsiRNAs, DNA including aptamers, and proteins including antibodies andantibody fragments. The invention also embraces candidate therapeuticwith different modes of action.

Candidate agents encompass numerous chemical classes, although typicallythey are organic compounds, proteins or antibodies (and fragmentsthereof that bind antigen). In some embodiments, the candidate agentsare small organic compounds, e.g., those having a molecular weight ofmore than 50 yet less than about 2500, for example less than about 1000and, in certain embodiments, less than about 500. Candidate agentscomprise functional chemical groups necessary for structuralinteractions with polypeptides and/or nucleic acids, and may include atleast an amine, carbonyl, hydroxyl, or carboxyl group, optionally atleast two of the functional chemical groups or at least three of thefunctional chemical groups. The candidate agents can comprise cycliccarbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfunctional groups. Candidate agents also can be biomolecules such asnucleic acids, polypeptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random or non-random polypeptides, combinatoriallibraries of proteins or antibodies, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc., to produce structural analogs ofthe agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc., which may be used to facilitate optimalprotein-protein and/or protein-agent binding. Such a reagent may alsoreduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

Kits

In some aspects of the invention, kits are provided. Kits of theinvention may contain nucleic acid, polypeptides, antibodies or othermolecules of the invention for use in vitro diagnosis, prognosis,monitoring of PML and/or JCV infection, exposure to JCV variants and/ortesting of candidate therapeutic agents. Components of the kits can bepackaged either in aqueous medium or in lyophilized form. Reagents foruse in PCR and ELISA assays may also be included in kits of theinvention as can detectable labeling agents in the form of intermediatesor as separate moieties to be conjugated as part of procedures to assayrisk for PML, JCV infection or exposure to a JCV variant. In someembodiments of a kit of the invention, the kit may include instructionsfor determining the presence of the variants of the invention and/or fordetermining the viral load of a JCV variant. The kit may also includecontrol values (e.g., reference numbers) that can be used forinterpreting results of methods used in the invention.

A kit of the invention may include nucleic acid, antibodies orpolypeptides that can be used to identify one or more indicia ofexposure to JCV variants having one or more PML-associated VP1 variants.In some embodiments, kits include materials for use in standardtechniques of ELISA to identify antibodies that can bind one or morepolypeptides comprising one or more variants indicative of exposure to aJCV variant. In some embodiments, a kit of the invention may includenucleic acid components for binding to and detecting nucleic acidsencoding one or more variants or variant combinations described hereinas associated with PML. In some embodiments, a kit may include one ormore different antibodies that specifically bind to one or morepolypeptides comprising one or more variants or combinations of variantsof the invention. A kit also may include components for use with theantibodies to determine expression of JCV variants or exposure to JCVvariants in a cell, tissue or subject.

A kit may comprise a carrier being compartmentalized to receive in closeconfinement therein one or more container means or series of containermeans such as test tubes, vials, flasks, bottles, syringes, or the like.The kit may also contain a control sample. In some embodiments, the kitcomprises instructions for interpreting test results such asinstructions for determining whether a test indicates whether the levelof a detected agent in the assay correlates with exposure to a JCVinfection (e.g., by a wild-type or variant JCV).

Biological Samples

Methods for assaying for exposure to a JCV variant may be carried out onany suitable biological sample. In some embodiments, a sample mayobtained from a subject and directly processed and assayed for indiciaof JCV as described herein. In some embodiments, cells may be isolatedfrom a biological sample and grown in culture prior to analysis. As usedherein, a subject may be a human or a non-human animal, including, butnot limited to a non-human primate, cow, horse, pig, sheep, goat, dog,cat, or rodent. Methods of the invention may be used to assay forexposure to a variant of JCV in subjects not yet diagnosed with PML.Methods of the invention may be used to assay for exposure to a JCVvariant in subjects not yet diagnosed as being infected with JCV. Inaddition, methods of the invention may be applied to subjects who havebeen diagnosed with PML and/or infection by a JCV variant. A sample maycomprise one or more cells. A sample may originate directly from asubject or from a cell culture. A sample may be processed (e.g., toprepare a cell lysate) or partially processed prior to use in methods ofthe invention. In some embodiments, a sample from a subject or culturemay be processed to obtain nucleic acids or polypeptides for use inassays to detect exposure to a variant of JC virus as described herein.Thus, an initial step in an assay may include isolation of a genomicnucleic acid sample and/or other nucleic acids and/or polypeptides froma cell, tissue, and/or other sample. Extraction of nucleic acids and/orpolypeptides may be by any suitable means, including routine methodsused by those of ordinary skill in the art such as methods that includethe use of detergent lysates, sonification, and/or vortexing with glassbeads, etc.

As used herein, the term “sample” means any animal material containingDNA or RNA or protein, such as, for example, tissue or fluid isolatedfrom an individual (including without limitation plasma, serum,cerebrospinal fluid, urine, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents. A sample containing nucleicacids may contain of deoxyribonucleic acids (DNA), ribonucleic acids(RNA), or copolymers of deoxyribonucleic acids and ribonucleic acids orcombinations thereof. A sample containing polypeptides may containpeptides and/or proteins. In some embodiments the sample containsantibodies. A sample may have been subject to purification (e.g.,extraction) and/or other treatment. The term “sample” may also refer toa “biological sample.”

As used herein, the term “biological sample” may refer to tissue, cellsor component parts (e.g., body fluids, including but not limited toblood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid,saliva, amniotic fluid, amniotic cord blood, urine, stool, vaginalfluid, and semen, etc.) of a subject. A “biological sample” may alsorefer to a homogenate, lysate, or extract prepared from tissues, cellsor component parts, or a fraction or portion thereof, including but notlimited to, for example, plasma, serum, spinal fluid, lymph fluid,urine, the external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, stool, milk, blood cells, tumors,or organs, CNS biopsies, etc.

Sample sources may include tissues, including, but not limited to lymphtissues; body fluids (e.g., blood, lymph fluid, etc.), cultured cells;cell lines; histological slides; tissue embedded in paraffin; etc. Theterm “tissue” as used herein refers to both localized and disseminatedcell populations including, but not limited to: brain, heart, serum,breast, colon, bladder, epidermis, skin, uterus, prostate, stomach,testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland,salivary gland, mammary gland, kidney, liver, intestine, spleen, thymus,bone marrow, trachea, and lung. Biological fluids include, but are notlimited to, blood, lymph fluid, cerebrospinal fluid, tears, saliva,urine, and feces, etc. Invasive and non-invasive techniques can be usedto obtain such samples and are well documented in the art. A controlsample may include a bodily fluid, a cell, a tissue, or a lysatethereof. In some embodiments, a control sample may be a sample from acell or subject that is free of PML and/or infection by, or exposure to,a JCV variant. In some embodiments, a control sample may be a samplethat is from a cell or subject that has PML and/or has been exposed to aJCV variant. In some embodiments a control sample is a sample comprisingwild-type JC virus.

Vaccines

The present invention further relates to a vaccine for immunizing amammal against PML or infection by a PML-associated JCV variant. Avaccine may comprise at least one polypeptide comprising one or more JCVvariant sequences of the invention. In some embodiments, a vaccine mayinclude one or more variant JCV or JCV peptides described herein aspredicted to reduce sialic acid binding. In some embodiments, a vaccinemay include a plurality of different peptides (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 10-15, 15-20, 20-50, or more) to provide a broad range ofdifferent epitopes. In some embodiments, the polypeptide is a fulllength JCV-VP1 variant polypeptide. In still another embodiment, thepolypeptide is a full length JCV-VP1 polypeptide selected from the groupconsisting of the polypeptides described herein (e.g., in FIG. 3 orTable 1 or 2B, Table 17 and/or Table 18, and/or one or more othervariants described herein), or any fragment thereof (e.g., a 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 100-150, 150-200 amino acid longpolypeptide, or a longer, shorter, or intermediate length polypeptide)having at least one variant amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more variant amino acids) at one or more of the predeterminedpositions described herein. In some embodiments, the polypeptide is afragment of a JCV-VP1 protein comprising one or more variant amino acidsof the invention. In a further aspect, the invention relates to apharmaceutical composition comprising at least one JCV variantpolypeptide and a suitable excipient, diluent or carrier. In someembodiments a polypeptide in the vaccine is about 5, about 10, about 20,about 30, about 40, or about 50 amino acids in length, or comprises thetotal length of the JCV-VP1 protein. These compositions are suitable forpreventing or treating PML and/or an infection by a JCV variantassociated with PML. A pharmaceutical composition may be administered toa subject in an effective amount to stimulate the production ofprotective antibody or protective T-cell response. In some embodiments,the vaccine is a nucleic acid vaccine comprising nucleic acids thatencode one or more variant JCV polypeptides of the invention.

The term “immunizing” refers to the ability of a substance to cause ahumoral and/or cellular response in a subject, whether alone or whenlinked to a carrier, in the presence or absence of an adjuvant, and alsorefers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. A PML “vaccine” is animmunogenic composition capable of eliciting protection against PML,whether partial or complete. A vaccine may also be useful for treating asubject having PML. Administration regimes for vaccines are known to aperson of ordinary skill in the art. In some embodiments, ranges ofamounts of polypeptide vaccines for prophylaxis of PML are from 0.01 to100 microgram/dose, for example 0.1 to 50 microgram/dose. Several dosesmay be needed per subject in order to achieve a sufficient immuneresponse and subsequent protection against PML or infection by a JCVvariant associated with PML.

Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers;and inactive virus particles. Such carriers are well known to those ofordinary skill in the art.

Adjuvants to enhance effectiveness of the polypeptide vaccines of theinvention include, but are not limited to: aluminum hydroxide (alum),N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S.Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE) and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of thethree components MPL, TDM or CWS may also be used alone or incombinations of two. In some embodiments, one or more peptides of theinvention may be used for immunization using one or more techniquesdescribed in Goldmann et al., 1999, Journal of Virology, Vol. 73, No. 5,pp 4465-4469, the disclosure of which is incorporated herein byreference.

In some embodiments, a vaccine can be administered to a subject prior toor along with the initiation of an immuno-suppressive therapy, or anyother treatment that may affect (e.g., weaken) the immune system.

Antibodies

In certain embodiments, antibodies or antigen-binding fragments thereofto JCV variants are also encompassed by the invention. Antibodies may beused in detection assays described herein (e.g., ELISA assays).Antibodies may be used in therapy to treat subjects with PML and/or toprevent or reduce infection by JCV variants. Suitable antibodies orfragments thereof may be selected for the ability to bind one or moreJCV variant polypeptides described herein. The antibody orantigen-binding fragment thereof may be an IgG1, IgG2, IgG3, IgG4, IgM,IgA1, IgA2, IgAsec, IgD, IgE or may have an immunoglobulin constantand/or variable domain of an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,IgAsec, IgD or IgE. In some embodiments, the antibody is a bispecific ormultispecific antibody. In further embodiments, the antibody is arecombinant antibody, a polyclonal antibody, a monoclonal antibody, ahumanized antibody or a chimeric antibody, or a mixture of these. Insome embodiments, the antibody is a human antibody, e.g., a humanmonoclonal antibody, polyclonal antibody or a mixture of monoclonal andpolyclonal antibodies. Antigen-binding fragments may include a Fabfragment, a F(ab)₂ fragment, and/or a Fv fragment CDR3.

Antibodies can be raised against a full length JCV-VP1 protein variantor against polypeptides variants comprising a partial sequence ofJCV-VP1 protein variant. Antibodies can be generated by injecting ananimal, for example a rabbit or goat or mouse, with the antigen (e.g., apolypeptide of a JCV-VP1 variant).

In order to prepare polyclonal antibodies, fusion proteins containing aJCV-VP1 variant can be synthesized in bacteria by expression ofcorresponding DNA sequences in a suitable cloning vehicle. The proteincan then be purified, coupled to a carrier protein and mixed withFreund's adjuvant (to help stimulate the antigenic response by therabbits) and injected into rabbits or other laboratory animals.Alternatively, the polypeptides can be isolated from cultured cellsexpressing the protein. Following booster injections at bi-weeklyintervals, the rabbits or other laboratory animals are then bled and thesera isolated. The sera can be used directly or purified prior to use,e.g., by methods such as affinity chromatography, Protein A-Sepharose,Antigen Sepharose, Anti-mouse-Ig-Sepharose. The sera can then be used toprobe protein extracts run on a polyacrylamide gel to identify the JCVvariant polypeptides. Alternatively, synthetic JCV variant polypeptidescan be made and used to inoculate animals.

To produce monoclonal JCV variant antibodies, mice are injected multipletimes (see above), the mice spleens are removed and resuspended in aphosphate buffered saline (PBS). The spleen cells serve as a source oflymphocytes, some of which produce antibodies of the appropriatespecificity. These are then fused with a permanently growing myelomapartner cell, and the products of the fusion are plated into a number oftissue culture wells in the presence of a selective agent such as HAT.The wells are then screened by ELISA to identify those containing cellsexpressing useful antibody. These are then freshly plated. After aperiod of growth, these wells are again screened to identifyantibody-producing cells. Several cloning procedures are carried outuntil over 90% of the wells contain single clones which are positive forantibody production. From this procedure a stable line of clones isestablished to produce the antibody. A monoclonal antibody can then bepurified by affinity chromatography using Protein A Sepharose,ion-exchange chromatography, as well as variations and combinations ofthese techniques (See e.g., U.S. Pat. No. 6,998,467).

In some embodiments, ‘humanized’ antibodies are used in therapy inhumans. Humanization of antibodies involves replacing native mousesequences with human sequences to lower the chance of an immune responseonce the therapeutic antibody is introduced into humans.

CNS Infections

JC polyomavirus is a virus that infects the brain and CNS (CentralNervous System). Subjects suffering from microbial infections of the CNSmay be susceptible to variant JC virus infection and/or proliferation inthe CNS. Microbial infections of the CNS may include, but are notlimited to, bacterial, fungal, protozoan, virus-like, and viralinfections. Infections include both acute and chronic conditions.Exemplary microbial CNS infections can result in a brain abscess,meningitis, encephalitis, vasculitis, or progressive multifocalleukoencephalopathy (PML). Most abscess-forming infections of the CNSare spread by the blood and are related to septicemia and endocarditis,although there may be direct infection that arises from sinus or middleear/mastoid infection. Bacterial infections of the CNS may include, butare not limited to infection by Streptococcus pneumonia, Streptococcuspyogenes, Staphylococcus aureu, Staphylococcus epidermidis,Enterobacteriacea, Propionibacterium, Pseudomonoas aeruginosa, Neisseriameningitis, Haemophilus influenzae or Listeria moncytogenes. Fungalinfections of the CNS are less common than bacterial infections, but mayarise in individuals with Acquired Immune Deficiency Syndrome (AIDS) andin other immunocompromised individuals, such as those undergoingchemotherapy or immunosuppressive therapy. An example of a protozoaninfection of the CNS is late-stage neurological trypanosomiasis, orsleeping sickness, which is caused by infection of the CNS bytrypanosoma protozoa.

Viral infections of the CNS may include, but are not limited to asepticmeningitis, encephalitis, and progressive multifocal leukoencephalopathy(PML). Acute neurological syndromes associated with viral infectioninclude, for example, acute viral encephalitis, flaccid paralysis,aspectic meningitis, and post infectious encephalomyelitis. Acute viralencephalitis may be caused by for example, herpes simplex virus,cytomegalovirus, varicella, rabies or an arbovirus. Common viral agentsof asceptic meningitis include, for example, enteroviruses, mumps virusand lymphocytic choriomeningitis virus. Post infectiousencephalomyelitis is a complication of infection with measles, mumps,rubella and primary varicella-zoster virus infection, for example.Guillain-barre syndrome is also an acute neurological syndromeassociated with viral infection.

Additional chronic neurological diseases attributable to viral infectioninclude, subacute sclerosing pan encephalitis (caused by persistentmeasles infection), spongiform encephalopathies (prion diseases) (e.g.,Creutzfeldt-Jakob disease (CJD), Gerstmann-Streussler Syndrome), andretroviral diseases (e.g., HIV-1 and HIV-2) characterized by paralysis,wasting, and ataxia.

Accordingly, aspects of the invention may be used to evaluate the riskof PML in subjects having one or more symptoms of a microbial CNSinfection.

Studies of JCV Variants

Both host and viral genetics may contribute to PML. Earlier studiesfocusing on viral genetic factors identified duplications andrearrangements in the regulatory region of the viral genome [Pfister LA, Letvin N L, Koralnik I J (2001) JC virus regulatory region tandemrepeats in plasma and central nervous system isolates correlate withpoor clinical outcome in patients with progressive multifocalleukoencephalopathy. J Virol 75: 5672-5676; Major E O, Amemiya K,Tornatore C S, Houff S A, Berger J R (1992) Pathogenesis and molecularbiology of progressive multifocal leukoencephalopathy, the JCvirus-induced demyelinating disease of the human brain. Clin MicrobiolRev 5: 49-73; Loeber G, Dorries K (1988) DNA rearrangements inorgan-specific variants of polyomavirus JC strain GS. J Virol 62:1730-1735; Martin J D, King D M, Slauch J M, Frisque R J (1985)Differences in regulatory sequences of naturally occurring JC virusvariants. J Virol 53: 306-311; and Zheng H Y, Takasaka T, Noda K,Kanazawa A, Mori H, et al. (2005) New sequence polymorphisms in theouter loops of the JC polyomavirus major capsid protein (VP1) possiblyassociated with progressive multifocal leukoencephalopathy. J Gen Virol86: 2035-2045]. Several studies with very limited sample numbers fromPML and healthy individuals also reported conflicting results onpossible association of several mutations in VP1 protein with PML [ZhengH Y, Takasaka T, Noda K, Kanazawa A, Mori H, et al. (2005) New sequencepolymorphisms in the outer loops of the JC polyomavirus major capsidprotein (VP1) possibly associated with progressive multifocalleukoencephalopathy. J Gen Virol 86: 2035-2045; Zheng H Y, Ikegaya H,Takasaka T, Matsushima-Ohno T, Sakurai M, et al. (2005) Characterizationof the VP1 loop mutations widespread among JC polyomavirus isolatesassociated with progressive multifocal leukoencephalopathy. BiochemBiophys Res Commun 333: 996-1002; Kato A, Sugimoto C, Zheng H Y,Kitamura T, Yogo Y (2000) Lack of disease-specific amino acid changes inthe viral proteins of JC virus isolates from the brain with progressivemultifocal leukoencephalopathy. Arch Virol 145: 2173-2182]. Nocomprehensive analysis of an association of changes in protein codinggenes of JCV with PML has been reported. Pathogenicity of virusesranging from influenza virus [Srinivasan A, Viswanathan K, Raman R,Chandrasekaran A, Raguram S, et al. (2008) Quantitative biochemicalrationale for differences in transmissibility of 1918 pandemic influenzaA viruses. Proc Natl Acad Sci USA 105: 2800-2805; and Chandrasekaran A,Srinivasan A, Raman R, Viswanathan K, Raguram S, et al. (2008) Glycantopology determines human adaptation of avian H5N1 virus hemagglutinin.Nat Biotechnol 26: 107-113] to the mouse polyomavirus [Bauer P H,Bronson R T, Fung S C, Freund R, Stehle T, et al. (1995) Genetic andstructural analysis of a virulence determinant in polyomavirus VP1. JVirol 69: 7925-7931; and Bauer P H, Cui C, Liu W R, Stehle T, Harrison SC, et al. (1999) Discrimination between sialic acid-containing receptorsand pseudoreceptors regulates polyomavirus spread in the mouse. J Virol73: 5826-5832], a close relative of human JCV, was shown to bedetermined by amino acid sequences involved in the binding of a viralcapsid protein to sialylated glycan receptors. Changes in the affinityand specificity of the virus for its cellular receptor(s) affect viralinfectivity and transmission, hence playing a crucial role in virulence.For example, a study of the mouse polyomavirus showed that VP1 aminoacid changes rather than changes in the non-coding regulatory region areresponsible for the increased pathogenicity of the virus.

Aspects of the invention are illustrated by experiments relating to theVP1 protein and its relationship to PML. Methods of molecular evolutionwere used to determine the presence of putative adaptive changes in theVP1 amino acid sequence associated with PML. The advantage of thisapproach over simple statistical association of sequence variants withthe disease, is that it takes into account the phylogenetic relationshipof viral strains and also allows identification of functionallysignificant amino acid positions by examining the rate of sequenceevolution.

According to aspects of the invention, a virus harboring substitutionsis adequately infectious if it was sufficiently abundant in the CNS ofPML patients to be isolated. In some embodiments, changes in glycanspecificity are predicted to allow JCV to lose its specificity tosialated glycans expressed outside of the CNS (e.g., RBCs). Thus, such avirus would avoid getting trapped on “pseudoreceptors” in the peripheryand travel unhindered from sites of viral shedding to enter the brain.Mutated virus must still maintain its specificity to glycans expressedon oligodendrocytes. This is consistent with the observation from themouse polyomavirus model where a mutation in a position orthologous toposition 269 of JCV affected viral ability to bind RBCs and also lead tothe dramatic increase in viral dissemination through the animal with alethal outcome [Dubensky T W, Freund R, Dawe C J, Benjamin T L (1991)Polyomavirus replication in mice: influences of VP1 type and route ofinoculation. J Virol 65: 342-349; and Freund R, Garcea R L, Sahli R,Benjamin T L (1991) A single-amino-acid substitution in polyomavirus VP1correlates with plaque size and hemagglutination behavior. J Virol 65:350-355]. Furthermore, there are several reports of JCV detection intonsils of many asymptomatically infected individuals [Kato A, KitamuraT, Takasaka T, Tominaga T, Ishikawa A, et al. (2004) Detection of thearchetypal regulatory region of JC virus from the tonsil tissue ofpatients with tonsillitis and tonsilar hypertrophy. J Neurovirol 10:244-249; and Monaco M C, Jensen P N, Hou J, Durham L C, Major E O (1998)Detection of JC virus DNA in human tonsil tissue: evidence for site ofinitial viral infection. J Virol 72: 9918-9923.]. Although thisobservation was taken as a support for the JCV infection of tonsilcells, it also could be explained by the viral trapping in lymphoidtissues. This is consistent with JCV binding to sialic acid in thetonsil tissue [Eash S, Tavares R, Stopa E G, Robbins S H, Brossay L, etal. (2004) Differential distribution of the JC virus receptor-typesialic acid in normal human tissues. Am J Pathol 164: 419-428].

According to the invention, given the large number of mutations that arespecific for PML, it is likely that more than a single mechanism (e.g.,two or more mechanisms) may play a role in PML etiology in different PMLcases. According to some aspects of the invention, PML associated VP1mutations may increase JCV tropism for brain white matter cells leadingto the increased viral infectivity and replication in oligodendrocytes.According to other aspects of the invention, mutations in PML may allowfor immune-escape by the virus. Out of the polyclonal immune responsedirected against the VP1 molecule only a limited number of antibodiesdirected against the cell receptor binding site (sialic acid) mayprovide protection against the spread of the viral infection. Mutationof an amino acid within an epitope crucial for the protective immunitymay allow virus to bind to its target cells and spread uninhibited.

Experiments described herein address how certain mutations occur in PMLand why, despite a very high prevalence of JCV, only a small proportionof immune deficient patients develop PML. According to aspects of theinvention, the absence of clustering of the mutations on the viralphylogenetic tree suggests that they arise independently in individualpatients rather than persist in the general populations as pathogenicviral variants. According to aspects of the invention, and based on theexperiments described herein, VP1 mutations play a very significant rolein the mechanism of PML emergence. Once a specific mutation affectingsialic acid binding occurs it allows virus to spread to the brain andinfect oligodendrocytes. The fact that the mutant virus was not detectedin the kidney suggests that that particular change in glycan bindingdoes not offer any selective advantage to the mutated virus in kidney.The mutations might have occurred and hence allowed the virus toestablish the residence in the brain under the conditions of immunesuppression shortly or long before the PML. Since no viral replicationwas detected in brains of asymptomatic individuals it is unlikely thatcompartmentalized evolution (e.g., intra CNS) prior to PML developmentcould account for the presence of mutated VP1 in CNS of PML patients.However, the issue of JCV latency in normal brain still remainscontroversial so it is still formally possible that non-mutated virushad entered the brain and mutations arose in the brain and notperiphery, e.g., kidney.

According to aspects of the invention, the healthy immune systemeffectively controls viral activation in the brain. However, as soon asthe immune system fails in certain individuals harboring such a mutatedvirus, the virus begins actively proliferating in oligodendrocytescausing PML. It is also possible that a healthy immune system mayefficiently suppresses newly developed mutants in their peripheral site(e.g., kidney) and prevent them from spreading and infecting new targetcells. Thus the timing of PML development may be mutation limited andthe interplay with environmental or host genetic factors may contributeto the non-deterministic development of PML. In addition, PMLdevelopment may be controlled by interactions of VP1 mutations withadditional genetic alterations of the virus including rearrangement ofthe viral regulatory region as it might give the virus additionalselective advantage in increasing viral replication in oligodendrocytes.

According to aspects of the invention, and as illustrated by theExamples described herein, JCV VP1 mutations affecting its receptorspecificity may be responsible for PML pathology. These results provideopportunities for the discovery of novel anti-polyomavirus therapeuticsand diagnostics of diseases caused by these viruses. The precise rolethat these mutations play in etiology of PML as well as how and wherethey arise requires further extensive investigation that would involveVP1 sequence analysis of longitudinal and time matching samples fromdifferent organs (e.g., urine, blood, CSF) and from a variety of PMLpatients.

The study of VP1 sequences from sequential samples, as described herein,showed the persistence of the same viral strains through the course ofdisease. Since the mutated virus represented the prevalent or solepopulation and it was maintained over time, it appears to be bothnecessary and sufficient for propagation of the infection. Notably,additional substitutions were acquired in case of relapse in a patientwho survived the first PML episode. This observation suggests, first,that the old mutated virus survives efficiently, either in the CNS or inthe periphery, and, second, that the emergence of a new substitution maytrigger a new PML episode. In some embodiments, a newly mutated virusmay arise in a context of virus activation under suboptimal immunecontrol and once it emerges, it may not be promptly recognized by theimmune system.

In some embodiments, aspects of the invention relate to the identity ofCSF and plasma VP1 sequences in PML patients. It appears that bothplasma-isolated and CSF-isolated virus are equally distinct fromurine-isolated virus, even if the mutation was excluded from theanalysis. This indicates that the CSF and plasma populations did notarise independently from urine but rather one of these populationsoriginated from the same source as the urine population and the otheroriginated from the first one. Although VP1 sequences carrying PMLgenicmutations were not observed in the urine of PML patients it is possiblethat that a mutation that was originally acquired during viralreplication in the kidney did not receive any competitive advantage overthe resident non-mutant virus to become a dominant or even a detectablepopulation in the kidney site. This is consistent with the observationthat VLPs prepared from most mutant VP1 molecules have lost the abilityto bind to the renal tubular epithelial cells, the site of viralinfection in the kidney.

According to aspects of the invention, without wishing to be limited bytheory, when a mutation causes a virus to lose it's specificity for oneor more widely occurring sialic acid containing receptors, the mutantvirus (in the blood circulation) is more likely than a wild-type virusto escape being trapped by a multitude of sialo-containingoligosacharide pseudoreceptors expressed on a great majority of cells inthe periphery. Accordingly, the mutant virus may be more likely to reachthe brain, or at least blood brain barrier (BBB). Once the virus managesto bypass peripheral obstacles and reaches the BBB it still has to crossthe endothelial cell layer to get to the CNS. In some embodiments, theextremely rare occurrence of PML, even in patients with significantimmune suppression, may be due to the required temporal constellation ofindependently rare events such as the appearance of certain mutant virusparticle and the presence of a BBB opening.

The hypothesis that mutations in VP1 protein contribute to PMLprogression by increasing viral chances of escaping peripheralcirculation to enter CNS is consistent with the observation that ˜10% ofall PML cases contained non-mutated virus. Thus even non-mutant virusmight also enter CNS given high level of viremia which could overwhelmthe peripheral defense of viral pseudoreceptors and also given thetemporally coincident opening in BBB. However, once a virus enters thebrain there might be no difference between mutant and non-mutant virusin their ability to infect oligodendrocytes and spread in CNS whichwould be consistent with retained ability of mutant VLPs to bind CNSderived astrocytes, another target of JCV during ongoing braininfection. Alternatively, VP1 mutation might have arisen during viralreplication in CNS as became dominant in CSF because it provided somecompetitive advantage for the virus to spread through the brain. Sincemost of the samples analyzed had all or >95% of all isolated clonescontaining mutations in order for the last hypothesis to be true themutations must have to occur very early during viral replication in thebrain. Since virus without mutations in VP1 can infect CNS cells quitesuccessfully mutations would not provided competitive advantage via gainof viral tropism to toward CNS cells although they might have improvedit. Still, the possibility that at least in some cases virus enters thebrain in non-mutated form and acquires the mutation during itsactivation within the CNS under the condition of immune suppression in apatient cannot formally be excluded.

Regardless of the site of selection, JCV VP1 substitutions appear to bekey for viral PML-genic potential. Viral capsid and envelope proteinsare critical to mediate viral attachment to viral target cells andinfectivity. Thus, mutations in influenza HA protein allow the virus tochange its subtle specificity for the sialic acid binding and ability ofthe virus to switch from zootropic to human infectivity. Similarly, someof these mutations were shown to be associated with increased influenzavirulence in human population as underscored by the example of 1918influenza by changing specificity from the sialic acid preferentiallyexpressed on cells in the upper portion of lung lobes to those expressedin lower portion of lung. Another mechanism by which a virus could gainan increase in virulence is probably via losing its wide receptorspecificity which causes virus to be trapped on cells that it does notinfect productively. One of the best studied examples of such amechanism comes from the studies of another polyomavirus, murinepolyomavirus infection in mice. It was demonstrated in this model that amutation at position 296, which is structurally orthologous to thecritical PMLgenic position 269 of JCV, dramatically changed viralspecificity for the sialic acids and affected viral ability to bindtarget cells and RBCs and also lead to the dramatic increase in viraldissemination through the animal resulting in a lethal outcome. Thus, itappears that a change in viral coat protein that affects viral bindingto its receptor is an extremely common mechanism that plays a crucialrole in altering viral pathogenesis and virulence with humanpolyomaviruses being no exception.

In some aspects, mutations at positions 55 and 269 to phenylanine aredetected in a sample from a subject. In other aspects, at least thesemutations are detected and indicate that the subject appeared to be mostcommon, suggesting that these 2 sites are under strong selectivepressure and provide JC virus with most advantage to cause PML. Thisobservation strongly correlates with the ability of mutations at thesetwo sites to abrogate viral binding to peripheral cells and sialic acidcontaining oligosaccharides thus suggesting that that particular loss offunction is most advantageous for the virus to be positively selectedmore frequently than other mutations. As described in more detailelsewhere herein, each of these sites accounted for 25% of all mutationsin the current study, with positions 267, 265 and 60 being next highestin frequency, each with 7.5% of all cases. Interestingly, CSF samplesfrom several patients contained two or more different viral populationseach carrying its own PML-specific mutation with no virus contained twosuch mutations simultaneously. Accordingly, some aspects of thisinvention provide that several different mutations may ariseindependently during normal viral replication and all might get selectedif each provides the virus the competitive advantage over non-mutatedvirus. In three cases, or ˜10% of all cases from this study, noPML-associated substitutions could be found in CSF and/or plasma. Othergenetic changes of JCV genome, e.g., involving mutations in the minorviral capsid proteins VP2 or VP3, might also be hypothesized in thesecases to explain the presence of these viruses in association with PML.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1 Detection of JCV Variants by PCR

Nucleic acids are isolated from a biological sample using establishedprotocols (e.g., cell lysis). Because the viral DNA may have integratedin the genomic DNA or may still be present as a smaller entity, bothgenomic DNA and shorter DNA sequences are isolated and subjected to PCRanalysis. Upon isolation the nucleic acids are resuspended in a bufferthat will facilitate PCR analysis. Buffers that facilitate PCR analysisare known to the skilled artisan (e.g., Maniatis) and are alsocommercially available from manufacturers of PCR enzymes (e.g., NewEngland Biolabs, Beverly, Mass.). Nucleotide primers are designed toresult in the amplification of the JCV-VP1 gene. PCR amplification is anestablished laboratory technique and comprises the addition ofnucleotide primers, a polymerase and single nucleotides, and polymerasebuffer and subjection this mixture to cycles of annealing, amplificationand dissociation resulting in the amplification of a desired DNAsequence. Upon amplification, the JCV-VP1 gene is separated from theresidual DNA and excess single nucleotides. The amplified JCV-VP1 DNA issequenced and the resulting nucleotide sequence is translated into apeptide sequence. This peptide sequence is subsequently compared to thevariant panels to determine which JCV-VP1 polypeptide variants arepresent in the biological sample.

Example 2 Detection of JCV Variants Using ELISA

Proteins and peptides are isolated from a biological sample usingstandard laboratory techniques (e.g., Maniatis). Both the cellularproteins and proteins of non-cellular components are subjected to theanalysis. In one assay the sample is interrogated for the presence ofJCV-VP1 polypeptides comprising one or more variants of the invention.The polypeptides are detected using sandwich ELISA comprising antibodiesspecific for JCV-VP1 polypeptides of the invention. The antibodies aregenerated by inoculating animals (e.g., rabbits) with the JCV-VP1polypeptides of the invention resulting in polyclonal antibodies. If sodesired, cells can be harvested from the inoculated animal to generatemonoclonal antibodies. Methods for the generation of both polyclonal andmonoclonal antibodies are routine in the art. The antibodies againstJCV-VP1 polypeptide variants are immobilized on a solid surface (e.g., a96-well plate), with one antibody type per well or surface area. Thebiological samples comprising the polypeptides are added to the wellsand incubated with the immobilized antibodies. Any polypeptide JCV-VP1variants present in the sample will bind to an antibody specific for thepolypeptide. After incubation, the sample is removed and the solidsurfaces are washed to remove any unbound material. As a next step, asolution containing additional antibodies specific for JCV-VP1 peptidesis added to the wells. This second aliquot of antibodies will create the“sandwich” (e.g., immobilized antibody: JCV-VP1 polypeptide: secondantibody). This second antibody can be detected using, for instance, alabeled tertiary antibody, allowing for the detection of JCV-VP1 variantpolypeptides. Alternatively, the secondary antibody itself may belabeled.

In a second ELISA assay, biological samples are assayed for the presenceof antibodies against one or more of the JCV-VP1 variant polypeptides.This assay can be use to determine whether a subject is currentlyinfected with, or has previously been exposed to, a JCV-VP1 variant.Even if the JCV-VP1 variant is no longer present, antibodies against thevariant may still be present in the biological sample and can bedetected. In this ELISA assay JCV-VP1 polypeptides are attached to asolid surface and the biological samples are incubated with thesepolypeptides. If antibodies specific for these polypeptides are presentin the biological samples they will bind to the polypeptides. Anyunbound material is again removed. The presence of bound antibody isdetected using a labeled secondary antibody.

Example 3 Determining Solvent Accessible Surface Area Composition of VP1Protein

Accessible surface area calculations require the knowledge of the 3Dcoordinates of biomolecule. A homology model of JCV VP1 virus-likeparticle was constructed using structure of CoAl of SV40 virus-likeparticle as a template (PDB ID: 1SVA). MODELER (A. Sali & T. L.Blundell. Comparative protein modelling by satisfaction of spatialrestraints. J Mol. Biol. 234, 779-815, 1993) algorithm was used formodel building and the SCWRL3 (A. A. Canutescu, A. A. Shelenkov, and R.L. Dunbrack, Jr. A graph theory algorithm for protein side-chainprediction. Protein Science 12, 2001-2014 (2003)) approach was used forside-chain position refinement. The polar and non-polar solventaccessible surface areas of amino acid sidechains were calculated usingLee and Richards's method (B. Lee B & F. M. Richards. The Interpretationof Protein Structures: Estimation of Static Accessibility. J. Mol. Biol55, 379-400 (1971)). Subsequently, 3D models of virus-like particles ofJCV VP1 variants were constructed, and polar and non-polar solventaccessible surface areas of their side-chains were subsequentlycalculated. Difference in polar surface area upon variant was calculatedby subtracting polar solvent accessible surface areas of the consensussequence from the polar solvent accessible surface areas of the varianton per amino acid side chain basis. Difference in non-polar surface areaupon variant was calculated by subtracting non-polar solvent accessiblesurface areas of the consensus sequence from the non-polar solventaccessible surface areas of the variant on per amino acid side chainbasis. The calculation results are presented in Table 3.

TABLE 3 Variant Gain in Non-Polar SA Gain in Polar SA L55−>F55 14.12 0K60−>M60 27.35 −13.22 K60−>N60 −10.18 −3.54 K60−>E60 −5.55 −2.1 S61−>L6149.72 −9.78 D66−>H66 25.86 −7.95 D66−>N66 15.35 −23.4 E69−>D69 −6.01−27.12 N74−>S74 −0.12 −30.62 K75−>R75 1.43 11.83 N265−>D265 −4.55 8.23N265−>T265 11.51 −12.15 S267−>F267 92.2 −33.04 S267−>L267 85.2 −31.3S269−>F269 75.44 −15.91 S269−>Y269 52.69 21 Q271−>H271 6.96 6.11

Example 4 Sequence Analysis of JCV Sequences

PML is a progressive and mostly fatal demyelinating disease caused by JCvirus infection and destruction of infected oligodendrocytes in multiplebrain foci of susceptible individuals. While JC virus is highlyprevalent in the human population, PML is a rare disease thatexclusively afflicts only a small percentage of immunocompromisedindividuals including those affected by HIV (AIDS) or immunosuppressivedrugs. Specific viral and/or host specific factors and not simply immunestatus must be at play to account for the very large discrepancy betweenviral prevalence and low disease incidence. According to the invention,several amino acids on the surface of the JC virus capsid protein VP1display accelerated evolution in viral sequences isolated from PMLpatients but not in sequences isolated from healthy subjects. Theexamples described herein provide strong evidence that at least some ofthese mutations are involved in binding of sialic acid, a known receptorfor the JC virus. Statistical methods of molecular evolution were usedto perform a comprehensive analysis of JC virus VP1 sequences isolatedfrom 55 PML patients and 253 sequences isolated from the urine ofhealthy individuals and found that a subset of amino acids foundexclusively among PML VP1 sequences is acquired via adaptive evolution.Modeling of the 3D structure of the JC virus capsid showed that theseresidues are located within the sialic acid binding site, a JC virusreceptor for cell infection. The involvement of some of these sites inreceptor binding was demonstrated by showing a profound reduction inhemagglutination properties of viral like particles made of the VP1protein carrying these mutations. All together these results indicatethat a more virulent PML causing phenotype of JC virus is acquired viaadaptive evolution that changes viral specificity for its cellularreceptor(s).

Methods:

35 full length VP1 sequences of JC viruses isolated from PML patientsand 253 full length VP1 sequences of JC viruses isolated from healthysubjects were downloaded from Genbank. In addition, 20 partial VP1sequences were available from Genbank enabling the analysis of the totalof 55 sequences for positions 43-287. In addition to these 55 VP1sequences isolated from PML patients Table 4 also contains informationfrom twelve more partial sequences available from a publication by Salaet al. [Sala M, Vartanian J P, Kousignian P, Delfraissy J F, Taoufik Y,et al. (2001) Progressive multifocal leukoencephalopathy in humanimmunodeficiency virus type 1-infected patients: absence of correlationbetween JC virus neurovirulence and polymorphisms in the transcriptionalcontrol region and the major capsid protein loci. J Gen Virol 82:899-907.]. It should be noted in these examples that all viral samplesisolated from PML patients originated from brain or CSF tissues exceptone sample isolated from kidney (Table 4). All viral samples isolatedfrom healthy subjects originated from urine. Multiple sequencealignments were constructed using TCoffee [Notredame C, Higgins D G,Hering a J (2000) T-Coffee: A novel method for fast and accuratemultiple sequence alignment. J Mol Biol 302: 205-217]. A number of PMLsequences were isolated from the same individual. In studying theevolution of viral sequences, same patient isolated sequences wereaccepted for the analysis as long as they differed from each other by ≧1nucleotide. However, identical “clonal” sequences were excluded from theanalysis. This resulted in the final set of 28 full-length VP1 sequencesand 42 partial VP1 sequences isolated from PML patients. All informationon the origin and clonality of sequences is contained in Table 4.Phylogenetic trees were built using the PhyMLmaximum likelihood method[Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm toestimate large phylogenies by maximum likelihood. Syst Biol 52: 696-704]with F84 substitution model [Kishino H, Hasegawa M (1989) Evaluation ofthe maximum likelihood estimate of the evolutionary tree topologies fromDNA sequence data, and the branching order in hominoidea. J Mol Evol 29:170-179 and Felsenstein J, Churchill G A (1996) A Hidden Markov Modelapproach to variation among sites in rate of evolution. Mol Biol Evol13: 93-104.] and using several methods included in the PHYLIP package(Felsenstein, J. 2005. PHYLIP version 3.6. Distributed by the author.Department of Genome Sciences, University of Washington, Seattle). VP1sequences isolated from PML patients and random subsets of sequencesisolated from healthy subjects were further analyzed using PAML [Yang Z(1997) PAML: a program package for phylogenetic analysis by maximumlikelihood. Comput Appl Biosci 13: 555-556]. Multiple models of sequenceevolution (M0-M8) were studied. A likelihood ratio test was used toevaluate the difference between models M1 and M2 to test for positiveselection. Residues with Bayes Empirical Bayes posterior probabilitiesexceeding 0.5 in the analysis of either full-length or partial set arereported in Table 5. Spidermonkey [Poon A F, Lewis F I, Frost S D,Kosakovsky Pond S L (2008) Spidermonkey: rapid detection of co-evolvingsites using Bayesian graphical models. Bioinformatics 24: 1949-1950] wasused to analyze epistatic interaction. Spidermonkey was run through theDatamonkey web server [Pond S L, Frost S D (2005) Datamonkey: rapiddetection of selective pressure on individual sites of codon alignments.Bioinformatics 21: 2531-2533].

TABLE 4 JCV VPl sequences from non-PML patients: BAC66394, BAC66418,BAC66382, BAB11716, BAB11722, AAK28466, AAK28460, AAK28478, BAC66400,BAC66388, BAD06126, BAC66406, AAK97970, AAK97964, BAC81952, BAC81958,AAM89309, AAM89303, BAC81922, BAC81916, BAC81910, BAC81904, AAM89297,BAD11896, BAC81946, BAE45426, BAE45360, BAD06120, BAE45432, BAA01962,BAE45420, BAE45414, BAE45384, BAE45378, BAE45372, AAK98036, BAE45402,BAE45408, BAE45396, BAE45444, BAD06024, BAE75838, BAE75832, BAE75826,BAE75820, BAE75814, AAK98030, AAK98024, AAK98018, AAK98010, AAK98006,AAK98000, BAE45438, BAE45390, BAD06108, BAD06102, BAD06096, BAD06090,BAD06084, BAD06048, BAD06030, BAD06018, BAD06054, BAD06042, BAD06036,AAG30857, BAE45366, AAN85455, BAD06150, AAN85449, AAK98042, BAD06174,BAD06156, BAD06072, BAD06060, AAN85473, BAC81840, BAF40841, BAF40835,BAF40829, BAF40823, BAF40811, BAF40847, BAF40781, BAF40817, BAF40799,BAF40793, BAF40787, BAF40745, AAN85467, AAN85461, BAC81834, BAF40751,BAF40805, BAA01961, BAD98972, BAD98966, BAD06227, BAC66430, BAC66412,BAD91887, BAD21235, BAD27118, BAC66424, BAA01958, BAB11710, BAD21265,BAD21259, BAD21253, BAD21241, BAD21229, BAD21247, BAD21283, BAD21271,BAD21295, BAD21289, BAA01959, BAA01960, BAD11848, BAD11842, AAM89339,AAM89327, BAD11836, BAC81852, BAC81858, BAD06144, AAG37198, AAM89315,BAD06138, BAD11890, BAD11884, BAD11878, BAD11872, BAD11866, AAK97994,BAB11698, BAC81940, BAC81964, AAK97946, BAD06066, BAF40769, BAC81870,BAC81864, BAC81934, BAC66376, BAC81874, BAC81846, AAK97940, BAC81898,BAC81892, AAK97922, AAK97916, AAK97910, AAK97982, BAF40763, BAD06078,AAK97958, BAD11860, BAF40757, BAD06162, AAM89321, BAD11854, AAK97928,BAD11830, BAF40775, BAB11704, BAC81928, AAK97988, BAD11902, BAD11824,BAD06233, BAC81886, BAC81880, AAM89345, BAD06168, AAM89333, BAD06132,BAC82365, AAK97952, BAA01964, BAA01963, AAK97934, BAD06114, AAK97976,BAD21277, AAR13077, BAE02908, AAR12957, AAR02463, AAR02457, BAE03058,AAR89235, BAE02896, AAR89241, BAE02890, BAE03064, BAE03070, BAE03082,AAG34673, AAG34667, AAR89205, AAR89217, AAR13659, BAE03088, BAE03160,AAQ88264, AAR89187, AAR89283, AAK28472, AAR06661, AAR89253, AAR89247,AAR89199, AAR89193, AAR89229, AAR89223, AAR89265, AAR32743, AAR89277,BAE03166, BAE02920, BAE02914, BAE03112, BAE03106, BAE03100, BAE03094,BAE03076, BAE02944, BAE02998, BAE02992, BAE02986, BAE02980, BAE02974,BAE02968, BAE02962, BAE03016, BAE02902, BAE03040, AAR89211, AAR89271,BAE02956, BAE02938, BAE02932, BAE03148, BAE02950, BAE03004, BAE03154,BAE03142, BAE03136, BAE03130, BAE03124, BAE03118, BAE02926. DNA ProteinDNA Isolate AA Start Patient N accession# accession # Source namelength, AA # 1 AF015537 AAB94036 brain 601 354 1 1 2 AB183539 BAE00111brain 1-1 354 1 2 3 AB183540 BAE00117 brain 1-2 354 1 2 4 AB183541BAE00123 brain 1-3 354 1 2 5 AB183542 BAE00129 brain 2-1 354 1 3 6AB183543 BAE00135 brain 2-2 354 1 3 7 AB183544 BAE00141 brain 2-3 354 13 8 AB190449 BAE00147 brain 3-1 354 1 4 9 AB190453 BAE00171 brain 3-5354 1 4 10 AB190452 BAE00165 brain 3-4 354 1 4 11 AB190451 BAE00159brain 3-3 354 1 4 12 AB190450 BAE00153 brain 3-2 354 1 4 13 AY536239AAT09819 CSF SA21_01 354 1 5 14 AB212952 BAE94726 brain ac-1 354 1 6 15AB212953 BAE94732 brain ac-2 354 1 7 16 D26589 BAA05636 brain Aic-1a 3541 8 17 AF004349 AAB62680 kidney GS/K 354 1 9 18 AF004350 AAB62687 brainGS/B 354 1 9 19 D11365 BAA01967 brain Her1-Br 354 1 10 20 AB214923BAE02848 CSF JVL-10 245 39 11 21 AB214924 BAE02849 CSF JVL-11 245 39 1222 AB214925 BAE02850 CSF JVL-12 245 39 13 23 AB214926 BAE02851 CSFJVL-13 245 39 14 24 AB214927 BAE02852 CSF JVL-16 245 39 15 25 AB214928BAE02853 CSF JVL-17 245 39 16 26 AB214929 BAE02854 CSF JVL-18 245 39 1727 AB214930 BAE02855 CSF JVL-19 245 39 18 28 AB214912 BAE02837 brainJVL-1a 245 39 19 29 AB214913 BAE02838 brain JVL-1b 245 39 19 30 AB214914BAE02839 brain JVL-1c 245 39 19 31 AB214915 BAE02840 brain JVL-1d 245 3919 32 AB214916 BAE02841 brain JVL-2 245 39 20 33 AB214931 BAE02856 CSFJVL-20 245 39 21 34 AB214917 BAE02842 brain JVL-3 245 39 22 35 BAE02843BAE02843 brain JVL-4 245 39 23 36 AB214919 BAE02844 brain JVL-5 245 3924 37 AB214920 BAE02845 brain JVL-7 245 39 25 38 AB214921 BAE02846 brainJVL-8 245 39 26 39 AB214922 BAE02847 brain JVL-9 245 39 27 40 J02226AAA82101 brain Mad-1 354 1 28 41 D11364 BAA01966 brain Mad11-Br 354 1 2942 D11363 BAA01965 brain Mad8-Br 354 1 30 43 D11366 BAA01968 brain NY-1B354 1 31 44 AB212954 BAE94738 brain oh-1 354 1 32 45 AY536243 AAT09843CSF SA27_03 354 1 33 46 AY536242 AAT09837 CSF SA28_03 354 1 34 47AY536241 AAT09831 CSF SA296_0 354 1 35 48 AY536240 AAT09825 CSF SA84_00354 1 36 49 D11367 BAA01969 brain Sap-1 354 1 37 50 D26590 BAA05637brain Tky-1 354 1 38 51 AB038254 BAB11728 brain Tky-1 354 1 39 52AB038255 BAB11734 brain Tky-2a 354 1 40 53 D26591 BAA05638 brain Tky-2a354 1 41 54 D11368 BAA01970 brain Tokyo-1 354 1 42 55 AF030085 AAC40846brain Tokyo-1? 354 1 43 56 U21840 AAB60586 brain 133 219 44 57 U21839AAB60584 brain 133 219 45 58 NA NA CSF P9VP1 136 11 46 59 NA NA CSFP8VP1 136 11 47 60 NA NA CSF P7VP1 136 11 48 61 NA NA CSF P5VP1 136 1149 62 NA NA CSF P4VP1 136 11 50 63 NA NA CSF P2VP1 136 11 51 64 NA NACSF P1VP1 136 11 52 65 NA NA CSF P12VP1 136 11 53 66 NA NA CSF P11VP174136 11 54 67 NA NA CSF P11VP173 136 11 54 68 NA NA CSF P11VP172 136 1154 69 NA NA CSF P10VP1 136 11 55

Results:

JCV VP1 gene sequences were downloaded from GenBank (Table 4) and usedto construct a phylogenetic tree for a random subset of sequencesisolated from healthy individual and full-length sequences isolated fromdistinct PML patients (FIG. 4 a). FIG. 4 is a phylogenetic distributionof PML associated viruses. (A) Broad phylogenetic distribution of PMLcausing JC viruses. Tree branches (labeled by G1 numbers) correspondingto PML causing viruses and viruses isolated from healthy subjects areindicated. The tree is constructed based on DNA sequences of VP1 geneusing maximum likelihood method. Only one sequence per patient wasincluded. (B) Phylogenetic distribution of mutations in the codon 269.The tree represents VP1 genes (labeled by G1 numbers) of virusesisolated from PML patients. Mutations in Ser269 codons are indicated bytext inserts. Circles on branches reflect aLRT support. Position 269 wasmasked prior to constructing the tree to avoid attraction of brancheswith mutations of this codon. The PhyML maximum likelihood method[Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm toestimate large phylogenies by maximum likelihood. Syst Biol 52:696-704.] was used with F84 substitution model [Kishino H, Hasegawa M(1989) Evaluation of the maximum likelihood estimate of the evolutionarytree topologies from DNA sequence data, and the branching order inhominoidea. J Mol Evol 29: 170-179 and Felsenstein J, Churchill G A(1996) A Hidden Markov Model approach to variation among sites in rateof evolution. Mol Biol Evol 13: 93-104]. Application of several methodsincorporated in the PHYLIP package maximum likelihood method,distance-based and parsimony-based methods of phylogeneticreconstruction produced similar results. Viral sequences isolated fromPML patients do not cluster on the phylogenetic tree and are broadlydistributed among viral types and geographic origins of the samples(FIG. 4 a). This is further supported by very low populationstratification measure F_(ST) [Slatkin M, Maddison W P (1990) Detectingisolation by distance using phylogenies of genes. Genetics 126: 249-260](1.8%). In agreement with earlier studies [Zheng H Y, Takasaka T, NodaK, Kanazawa A, Mori H, et al. (2005) New sequence polymorphisms in theouter loops of the JC polyomavirus major capsid protein (VP1) possiblyassociated with progressive multifocal leukoencephalopathy. J Gen Virol86: 2035-2045, Jobes D V, Chima S C, Ryschkewitsch C F, Stoner G L(1998) Phylogenetic analysis of 22 complete genomes of the humanpolyomavirus JC virus. J Gen Virol 79 (Pt 10): 2491-2498, and Agostini HT, Deckhut A, Jobes D V, Girones R, Schlunck G, et al. (2001) Genotypesof JC virus in East, Central and Southwest Europe. J Gen Virol 82:1221-1331], PML causing viruses are not limited to a specific viralphylogenetic type.

Sequences from viruses isolated from PML patients were used as well asthose from healthy subjects with the goal of determining whether PMLassociated evolutionary selective pressure is acting on the viral VP1gene. This analysis utilized the PAML package [Yang Z (1997) PAML: aprogram package for phylogenetic analysis by maximum likelihood. ComputAppl Biosci 13: 555-556] designed to identify the presence of codonsevolving under positive selection. PAML evaluates multiple evolutionarymodels using the parametric likelihood ratio test. Several models weretested including a model of neutral evolution, a nearly neutral modelallowing for purifying (e.g., negative) selection, and a heterogeneousmodel that allows some codon positions to evolve under positiveselection and other codon positions to evolve under negative selectionor neutrally (Table 5). A number of more complex models also weretested.

In the case of VP1 sequences from JCV isolated from healthy subjects,the nearly neutral evolutionary model involving a mixture of neutrallyevolving codons and codons under purifying selection clearlyoutperformed the purely neutral model (p-value 7.0×10⁻⁶). However, nostatistical support was found for more complex models including modelswith positive selection. In contrast, for VP1 sequences isolated fromPML patients, allowing codons to evolve under positive selectionresulted in a highly significant increase in the model likelihood (Table5). The model with three categories of sites including sites evolvingunder purifying selection, neutral sites and sites under positiveselection explained the data significantly better than the nearlyneutral model limited only to neutral sites and the sites underpurifying selection (p-value 2.5×10⁻⁷). More complex models did not showsignificant improvement over the simplest model with three categories ofcodons.

Four codon positions (corresponding to amino acids 55, 60, 267 and 269)were identified as evolving under positive selection in the PML samplingof full length sequences (Table 5). Bayesian posterior probabilities forpositive selection computed by PAML were above 0.5 for these codonpositions. The posterior probability for positive selection in codon 269was close to 1. To increase the power of analysis, partial VP1 sequenceswere added from JC virus isolated from PML patients. The addition ofpartial sequences revealed signal of positive selection in codon 265(Table 5).

TABLE 5 Codons under positive selection in the PML sample. Full lengthsequence Partial sequence set set (n = 28) codons 43-287 (n = 42)P-value for the positive selection test Mutations 2.5 × 10⁻⁷ 3.5 × 10⁻⁶Position WT Mutant Bayes Empirical Bayes posterior probability 55 L F0.82 0.94 60 K M, E, N <0.5 0.94 265 N D, T <0.5 0.85 267 S F, L 0.800.92 269 S F, Y, C 1.00 1.00

In this example, two VP1 mutations were not observed in the same JCVisolate. Analysis by the Spidermonkey [Poon A F, Lewis F I, Frost S D,Kosakovsky Pond S L (2008) Spidermonkey: rapid detection of co-evolvingsites using Bayesian graphical models. Bioinformatics 24: 1949-1950]method revealed epistatic interactions between positions 55 and 269 andbetween position 60 and 269 (with posterior probabilities 0.88 and 0.70respectively). This may reflect “diminishing return” epistaticinteractions, e.g., subsequent mutations are not beneficial and possiblydetrimental on the background of a single mutation.

All substitutions in these five codons are clearly associated with PML.At least 52% of JC viruses (or 36 out of 69 sequences, including partialsequences) isolated from PML patients have at least one of thesemutations, whereas none of these substitutions have been observed in 253full length viral sequences from healthy subjects (Table 6).

TABLE 6 Amino acid variability of JCV VP1 sequences

Residues highlighted with darker shading are distinct between PML andnon-PML groups and have Bayes Empirical Bayes posterior probability forpositive selection >0.5 (Table 5). Residues highlighted with lightershading are distinct between PML and non-PML groups.

The strongest signal of positive selection in the PML sample wasdetected for the codon encoding amino acid at position 269. FIG. 4 bshows that multiple independent mutations of Ser269 to aromatic residuesphenylalanine and tyrosine were observed in VP1 from PML associatedviruses. The existence of multiple independent mutations is not anartifact of phylogenetic reconstruction because lineages with mutantvariants are separated by multiple branches with over 90% support bybootstrap analysis and support of the likelihood ratio test implementedin PhyML [Guindon S, Gascuel O (2003) A simple, fast, and accuratealgorithm to estimate large phylogenies by maximum likelihood. Syst Biol52: 696-704]. These lineages correspond to different, previouslyidentified, phylogenetic types of JC virus and are from diversegeographic locations [Jobes D V, Chima S C, Ryschkewitsch C F, Stoner GL (1998) Phylogenetic analysis of 22 complete genomes of the humanpolyomavirus JC virus. J Gen Virol 79 (Pt 10): 2491-2498 and Agostini HT, Deckhut A, Jobes D V, Girones R, Schlunck G, et al. (2001) Genotypesof JC virus in East, Central and Southwest Europe. J Gen Virol 82:1221-1331].

VP1 sequences isolated from PML patients and random subsets of sequencesisolated from healthy subjects were further analyzed using PAML [Yang Z(1997) PAML: a program package for phylogenetic analysis by maximumlikelihood. Comput Appl Biosci 13: 555-556].

Multiple models of sequence evolution incorporated in PAML were examinedincluding purely neutral model (M0), nearly neutral model (M1), modelwith positive selection (M2) and additional more complex models (M3-M8).A likelihood ratio test (LRT) was used to compare the difference betweenmodels M1 and M2 to test for positive selection. P-values for positiveselection in three datasets are shown together with Bayesian posteriorprobabilities for each codon position. Residues with Bayes EmpiricalBayes posterior probabilities exceeding 0.5 are shown.

Example 5 Identified Mutations Fall in the Sialic Acid Binding SiteMethods:

A homology model of the JCV VP1 protein pentameric unit was built withMODELER [Sali A, Blundell T L (1993) Comparative protein modelling bysatisfaction of spatial restraints. J Mol Biol 234: 779-815] using thestructure of MPyV VP1 (Protein Data Bank ID: 1VPS [Stehle T, Harrison SC (1997) High-resolution structure of a polyomavirus VP1-oligosaccharidecomplex: implications for assembly and receptor binding. Embo J 16:5139-5148] as a template. The model ofNeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc tetrasaccharide wasbuild based on the structure ofNeuNAc-(α2,3)-Gal-(β1,3)-[(α-2,6)-NeuNAc]-Glc-NAc bound to MPyV VP1[Stehle T, Harrison S C (1997) High-resolution structure of apolyomavirus VP1-oligosaccharide complex: implications for assembly andreceptor binding. Embo J 16: 5139-5148]. The model of the JCVVP1/NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc tetrasaccharide wasextensively refined in CHARMM [Brooks B R, Bruccoleri R E, Olafson B D,States D J, Swaminathan S, et al. (1983) CHARMM: A program formacromolecular energy, minimization, and dynamics calculations. Journalof Computational Chemistry 4: 187-217] and was analyzed using PyMOLvisualization software (The PyMOL Molecular Graphics System (2002)DeLano Scientific, Palo Alto, Calif., USA. www.pymol.org).

Results:

According to aspects of the invention, the functional role of the fiveidentified amino acid positions can be evaluated by constructing athree-dimensional molecular model of the JC virus VP1 bound toNeuNAc-(α-2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc tetrasaccharide basedon the crystal structure of MPyV VP1/oligosaccharide complex [Stehle T,Harrison S C (1997) High-resolution structure of a polyomavirusVP1-oligosaccharide complex: implications for assembly and receptorbinding. Embo J 16: 5139-5148]. The structural model shown in FIG. 5 asuggests that all PAML-identified amino acids are clustered on thesurface of the VP1 protein at the sialic acid binding site and arelikely to be involved in sialic acid binding. FIG. 5 is a structuralmodel of JCV VP1/NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NActetrasaccharide complex. (A) A model of JCV VP1 basic pentamer incomplex with NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NActetrasaccharide. Surfaces of five chains of JCV VP1 are shown. The RGmotif essential for binding of core sialic acid is shown. PML-associatedmutated residues confirmed by PAML are indicated (L55, K60, S265, S267,S269). Additional mutations unique to PML-isolated samples also areshown (S61, D66, S123, H129, V223 and Q271). (B) A close-up view ofNeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc tetrasaccharide/JCV VP1complex. The location of V296 of MPyV VP1 which is predicted to beequivalent to S269 of JCV VP1 is shown in mesh. Additionally, in someembodiments the L55F, K60M, S267F, and S269F substitutions may inducesteric clashes with the modeled saccharide leading to a decrease in theaffinity of the interaction. Affinity to sialic acid was related toviral pathogenicity in multiple studies of flu virus, mousepolyomavirus, and mouse minute virus [Srinivasan A, Viswanathan K, RamanR, Chandrasekaran A, Raguram S, et al. (2008) Quantitative biochemicalrationale for differences in transmissibility of 1918 pandemic influenzaA viruses. Proc Natl Acad Sci USA 105: 2800-2805, Bauer P H, Cui C, LiuW R, Stehle T., Harrison S C, et al. (1999) Discrimination betweensialic acid-containing receptors and pseudoreceptors regulatespolyomavirus spread in the mouse. J Virol 73: 5826-5832 and Nam H J,Gurda-Whitaker B, Gan W Y, Ilaria S, McKenna R, et al. (2006)Identification of the sialic acid structures recognized by minute virusof mice and the role of binding affinity in virulence adaptation. J BiolChem 281: 25670-25677. Particularly, pathogenicity of mousepolyomavirus, a close relative of the JC virus, was mapped to a VP1amino acid substitution at position 296 [Bauer P H, Bronson R T, Fung SC, Freund R, Stehle T, et al. (1995) Genetic and structural analysis ofa virulence determinant in polyomavirus VP1. J Virol 69: 7925-7931], aposition orthologous to position 269 in human JC virus that showed thestrongest signal of positive selection in PML-causing viral isolates inthis study. As shown in FIG. 5 b, serine 269 of the human JC virus andvaline 296 of the mouse polyomavirus occupy identical locations in thesialic acid binding pocket.

Positions 61, 66, 123, 129, 223 and 271 are all limited to the PMLsample (Table 6) and also line up with the sialic acid binding pocket(FIG. 5 b). It is possible that those residues went undetected by thePAML analysis due to the small sample size and that the development ofPML is accompanied by positive selection for amino acids involved insialic acid binding in a majority of cases. The length of thephylogenetic tree in the analysis is short thus limiting the power todetect positive selection [Anisimova M, Bielawski J P, Yang Z (2001)Accuracy and power of the likelihood ratio test in detecting adaptivemolecular evolution. Mol Biol Evol 18: 1585-1592 and Anisimova M,Bielawski J P, Yang Z (2002) Accuracy and power of bayes prediction ofamino acid sites under positive selection. Mol Biol Evol 19: 950-958].Likelihood ratio test for detecting positive selection using a shorttree is conservative [Anisimova M, Bielawski J P, Yang Z (2001) Accuracyand power of the likelihood ratio test in detecting adaptive molecularevolution. Mol Biol Evol 18: 1585-1592], and Bayes Empirical Bayesanalysis is of limited power [Anisimova M, Bielawski J P, Yang Z (2002)Accuracy and power of bayes prediction of amino acid sites underpositive selection. Mol Biol Evol 19: 950-958]. Thus, additionalPML-specific VP1 mutations can also be positively selected. Mutations atresidue 107 are also found exclusively in the PML sample. However, itdid not show evidence of positive selection according to PAML and is notlocated in the sialic acid binding pocket.

Example 6 JCV Mutants and Sialic Acid Binding Methods: HemagglutinationAssay and Viral Like Particles

Hemagglutination assay was performed as previously described [ChapagainM L, Nguyen T, Bui T, Verma S, Nerurkar V R (2006) Comparison ofreal-time PCR and hemagglutination assay for quantitation of humanpolyomavirus JC. Virol J 3: 3 and Padgett B L, Walker D L (1973)Prevalence of antibodies in human sera against JC virus, an isolate froma case of progressive multifocal leukoencephalopathy. J Infect Dis 127:467-470]. Briefly, human type O blood was washed twice and suspended inAlsever's buffer (20 mM sodium citrate, 72 mM NaCl, 100 mM glucose, pH6.5 adjusted with acetic acid) at a final concentration of ˜0.5%. Serialtwo-fold dilutions of VLPs were prepared in Alsever's buffer and anequal volume of RBCs was added into each well of a 96-well “U” bottommicrotiter plate and incubated at 4° C. for 3-6 hr. Minimum HAconcentration is the lowest concentration of VLP protein that stillagglutinated RBCs.

Genes encoding the VP1 protein from JC virus strains BAE00117, AAT09831and AAQ88264 were created synthetically and cloned into the GatewaypDEST8 (Invitrogen) shuttle vector for transfer into the pFASTBACbaculovirus expression system for baculovirus expression in SF9 cells.Purification of VLPs was performed from roughly 100 grams of frozen cellpellets from 5 liters of culture. Cells were resuspended in 500 ml ofPBS containing 0.1 mM CaCl₂. The cells are disrupted by passing the cellsuspension twice through a Microfluidics Microfluidizer. Cell debris wasremoved by pelleting at 8000×G for 15 minutes. The supernatant volumewas adjusted to 720 ml with PBS/CaCl₂ and loaded onto 5 ml 40% sucrosecushions. Virus-like particles were twice pelleted through the sucrosecushions in a SW28 rotor at 100,000×G for 5 hours. The VLP pellets wereresuspended in PBS/CaCl₂ and then treated with 0.25% deoxycholate for 1hour at 37° C. followed by the addition of 4M NaCl/0.1 mM CaCl₂ for 1hour at 4° C. Precipitated material was removed by centrifugation at8000×G for 15 minutes. The resulting supernatant was concentrated andbuffer exchanged by ultrafiltration through a Pelicon-2 500,000 MWCOmembrane (Millipore). The concentrated VLPs were applied to the centerof a 25-40% step gradient of Optiprep (Sigma) and banded at 190,000 gfor 17 hours in a type 50.2 rotor. VLP bands were collected and thenconcentrated and buffer exchanged in an Amicon stirred cell with a300,000 MWCO membrane. VLP quality was determined by gel electrophoresisand electron microscopy (FIG. 6). Protein concentration was determinedby the Micro BCA assay (Pierce). Electron microscopy was performed atthe Department of Cell Biology at Harvard Medical School. VLP sampleswere placed on carbon grids, briefly washed in water and negativelystained with uranyl acetate and allowed to dry. The grids were viewedand imaged on a Technai G2 Spirit BioTWIN TEM.

Results:

In order to experimentally verify the role that these substitutions playin sialic acid binding by the VP1 capsid, viral like particles (VLP)were recombinantly produced from VP1 protein encoded by severaldifferent naturally occurring viruses. VLPs were generated from viralVP1 sequences encoding substitutions with one of the two strongestsignals of positive selection identified by PAML, one with phenylalanineat position 269 (F269) and another one with phenylalanine at position 55(F55). Two different VP1 genes that were used as controls do not harborany of the identified PML-associated mutations, one from a healthyindividual (WT) and another one from a PML patient (Mad-1) (Table 7).

TABLE 7 Amino acid variability of JCV VP1 sequences between VLPs. 55 7475 117 128 134 158 164 269 321 332 345 WT1 L N K T T A V T S I Q RAAQ88264 55F F N K T T A V T S I Q R AAT09831 WT2(Mad-1) L N R S T G L KS V E K P03089 269F L S K S A G V K F V E K BAE00117

Viral hemagglutination of red blood cells (RBCs) has been shown to be areliable measure of sialic acid binding by polyomaviruses [Freund R,Garcea R L, Sahli R, Benjamin T L (1991) A single-amino-acidsubstitution in polyomavirus VP1 correlates with plaque size andhemagglutination behavior. J Virol 65: 350-355 and Liu C K, Wei G,Atwood W J (1998) Infection of glial cells by the human polyomavirus JCis mediated by an N-linked glycoprotein containing terminalalpha(2-6)-linked sialic acids. J Virol 72: 4643-4649]. All four VLPswere tested in a hemagglutination assay. Strikingly, both F55 and F269variants displayed more than 8000-fold lower HA activity than eithercontrol VLP (Table 7A). Specifically, the F55 variant completely failedto agglutinate human type O RBCs even at 200 μg/ml, the highestconcentration tested, and the F269 variant displayed very low HAactivity as it caused hemagglutination only at concentrations above 25μg/ml. At the same time both L55 and 5269 carrying variants (WT andMad-1) caused hemagglutination of RBCs at concentrations down to 0.375ng/ml and 6.25 ng/ml, correspondingly. In this example, the F55 mutanthas the single amino acid difference with its corresponding wild typevariant (WT). Therefore the change in hemagglutination can bespecifically attributed to this amino acid replacement. In addition tothe change in position 269 the F269 mutant variant has two additionalamino acid positions that are different from its corresponding controlvariant (Mad-1). Both of those amino acid changes are not PML specificand are unlikely to explain the difference in hemagglutination. Whilethe Mad-1 isolate had originated from a PML patient [Padgett B L, WalkerDL, ZuRhein G M, Eckroade R J, Dessel B H (1971) Cultivation ofpapova-like virus from human brain with progressive multifocalleucoencephalopathy. Lancet 1: 1257-1260] it does not contain any of thePML-specific mutation which correlates well with its ability tohemagglutinate RBCs. The lack of PML-genic mutations in this PML isolatesuggests that VP1 mutations are not an exclusive mechanism leading toPML development.

TABLE 7A Residues 55 and 269 in VP1 protein play very important role inhemagglutination of RBCs by Viral Like Particles (VLPs). Viral variantMinimum HA VLP concentration, ng/ml WT1 0.08 55F >200,000 WT2 (Mad-1)6.25 269F 50,000

Hemagglutination was conducted as described in Materials and Methodsusing serial dilutions of VLPs starting from 200 μg/ml. VLPs were addedto type O RBC and incubated at 4° C. for 3 hours. Agglutination isvisualized by the lack of a round pellet formed by the settling of RBCsout of suspension. E55 is a VP1 variant with phenylalanine at theposition 55 (AAT09831), F269 is a VP1 variant with phenylalanine at theposition 269 (BAE0011). WT (AAQ88264) and Mad-1 (P03089) are VP1variants with leucine and serine at positions 55 and 269 respectively.

Protein sequences were aligned using ClustalW, amino acids different inat least one sequence from the rest of sequences are shown with theirpositions indicated.

Example 7 Mutant JCVs Have Impaired Binding to Gangliosides GangliosideELISA:

The following gangliosides were prepared at 1 mg/ml in methanol: asialoGM1 (human), monosialo GM1 (human), GM2 (human), GM3 (bovine), GM4(human), disialo GD1a (bovine), GD1b (human), GD2 (human), GD3 (bovine),trisialo GT1b (bovine). All gangliosides purchased from Calbiochemexcept GM2 which was purchased from American RadioChemicals.

Gangliosides were diluted to 0.1 mg/ml in methanol and added 0.1 ml/wellof an ELISA plate (Corning 9018); the methanol was allowed to evaporateovernight. On the following day, the plates were blocked with 0.25ml/well 1×PBS with Ca²⁺ and Mg^(2+, 1)% BSA (Fraction V), 0.1% Tween-20for one hour. The plates were then transferred onto ice—allconsequential incubations were performed at +4° C.

VLPs were prepared at 0.03 mg/ml in block buffer; the buffer was removedfrom the plate and the VLPs added 0.1 ml/well. The plate was incubatedon ice for 60-90 minutes. Anti-JCV VP1 (clone PAB597) was prepared at0.002 mg/ml in block buffer. The plates were washed with 0.25 ml/wellblock buffer; anti-VP1 was added 0.1 ml/well. The plate was incubated onice for 45-60 minutes. HRP-conjugated goat anti-mouse IgG (H+L) (JacksonImmunoResearch) was prepared at 1:5,000 in block buffer. The plate waswashed; 0.1 ml/well of HRP-anti-mouse was added. The plate was incubatedon ice for 30 minutes. The plate was washed and developed with 0.1 ml1-Step Turbo TMB (Thermo Scientific Pierce). Color development wasmonitored and the reaction stopped with addition 0.1 ml 2NH₃PO₄. Theplates were then read at 450 nm on spectromphotometer (MolecularDevices).

Each ganglioside had control wells (HRP-anti-mouse IgG only, PAB597 andHRP-anti-mouse IgG). The background control was designated asHRP-anti-mouse IgG only. Each experimental well was calculated using theformula (Experimental-Background)/Background.

The binding of WT JCV was evaluated against a variety of gangliosides.FIG. 7 shows that WT JCV binds to some glycans, but not to all. Thestructure of selected gangliosides is shown in FIG. 8 and their abilityto bind to WT JCV is indicated.

FIG. 9 shows that the F55 and F269 mutations are not capable of bindingNeu5Acα(2-3) and α(2-6) glycans. The structure of selected gangliosidesis shown in FIG. 10 and their ability to bind to mutant JCV isindicated.

FIG. 11 compares the ability of WT and mutant to JCV to bind selectedgangliosides.

Example 8 Mutant JCV Binds Glial Cell Lines but not Lymphocytes FlowCytometry Analysis of VLP Staining;

The following cells were used: SVG-A (gift from Walter Atwood), isolatedperipheral mononuclear cells from donors. Adherent cells were detachedusing Accutase, collected, and washed. Venous blood was drawn fromhealthy donors; PMBCs were isolated using a standard protocol involvingcentrifugation over Ficoll-Hypaque Plus (Amersham Biosciences).

All stainings were performed on ice in PBS buffer containing calcium andmagnesium, 1% bovine serum Albumin (fraction V), 2 mM sodium azide.

Cells (1-5×10⁵ cells/sample) were incubated with 10 μg/ml VLP diluted inFACS buffer, in a total volume of 0.05 ml, on ice for 60-90 minutes in96 V-bottom well plate. Cells were washed with 0.15 ml FACS buffer andcentrifuged at 2000 rpm (˜800×g) for 5 minutes. VLP binding was detectedby staining cells with anti-JCV VP1 (clone PAB597) at 0.002 mg/ml in0.05 ml for 45-60 minutes, followed by a wash step and detection withAlexa Fluor 488 anti-mouse IgG (H+ L) (Invitrogen) diluted 1:100 in 0.05ml/sample for an additional 30-45 minutes. Cells were washed and fixedin 0.05 ml Cytofix/Cytoperm (BD) for 15-25 minutes, washed andresuspended in 0.2 ml FACS buffer. The samples were analyzed on a FACSCalibur.

Mutant JCV (269F) does not bind lymphocytes. However, mutant JVC isstill capable of binding glial cell lines (FIG. 12). WT JCV and anegative control are also depicted.

Example 9 Generation of Mutant Specific Anti-JCV Antibodies

For the immunization of rabbits to generate anti-JCV VP1 sera, 0.5 mg ofVP1 protein in the form of virus like particles (VLPs) in PBS buffer wasinjected subcutaneously into 10 spots on the rabbit back (0.05 mg/spot).The primary immunization was followed by 2 boosts at 2-week intervalsafter which, sera was collected and assayed for anti-VP1 activity.

Detection of Anti-Mutant-JCV Antibody by Competition ELISA:

A: Anti-serum from 269F-VLP immunized rabbit or anti-WT-MAD1-immunizedVLP rabbit serum were pre-incubated with or without 100 ug/ml ofWT-MAD1-VLP, and were then incubated with a plate coated with 269F-VLP.Bound antibodies were revealed with a peroxidase-conjugated anti-rabbit.

B: Anti-serum from 55F-VLP immunized rabbit or anti-WT-immunized VLPrabbit serum were pre-incubated with or without 100 ug/ml of WT-VLP, andwere then incubated with a plate coated with 55F-VLP. Bound antibodieswere revealed with a peroxidase-conjugated anti-rabbit.

Rabbits were injected with F269 and F55 JCV mutant VP1 VLP resulting inthe generation of antibodies specific for mutant JCVs. A competitionELISA showed that the mutant specific JCV antibodies can bedistinguished from a WT JCV antibody (FIG. 13). The assay configurationis illustrated in FIG. 14. Antibodies bind mutant VLP (either F55 orF269) captured on the plate. Competition is done with non-mutant VLP toabsorb all antibodies directed at “backbone” of the molecule, leavingonly antibodies against mutant epitopes (if such are present in thesample) to bind to the plate and be detected.

The mutant JCV and WT JCV antibodies were also compared for theirability to bind to a mutant JCV-VLP. ELISA plates were coated with F269and F55 mutant JCV polypeptides and WT JCV and mutant JCV antibodieswere added to the plates (FIGS. 15 and 16). Both the mutants and WT JCVantibodies bind to the coated ELISA plate. Addition of WT JCV (JCV-471and JCV-MAD1) resulted in the disappearance of the binding of the WT JCVantibody, while the mutant antibodies remain bound to the ELISA plate.Furthermore addition of JCV virus resulted in the disappearance of thebound WT antibody, while the mutant antibody remains bound to the plate.

Example 10 JCV-VP1 Mutations as a Viral Immune Escape Mechanism in SomePatients

Serum from a patient that carries 269F mutation in VP1 protein oranti-WT-MAD1-immunized VLP rabbit serum (as a positive control) werepre-incubated with or without 100 μg/ml of WT-MAD1-VLP, and were thenincubated with a plate coated with 269F-VLP. Bound antibodies wererevealed with a peroxidase-conjugated anti-human or rabbit antibodies todetect antibody binding to the coated 269F mutant VLP.

FIG. 17 shows that a patient that has the F269 mutant virus hasdeveloped an antibody response against the WT virus but not against theF269 mutant virus.

FIG. 18 shows that (top) rabbit immunized with non-mutant VLP raisesantibody to the site(S269 in this cases) that could get mutated. In thiscases experiment was done similar to schematics on slide 16 (aboveinsert) with several differences, instead of the mutant VLP a non-mutantVLP was coated on the plate and antibody binding in sera was competedwith mutant (either F55 or F269) protein), so only antibody tonon-mutated AA epitope would be left to bind to the plate. (bottom)shows the same experiment with healthy volunteer sample. The results inTable 8 are based on a similar experiment with several differentvolunteer samples. Table 8 shows that people that are not suffering fromPML can have antibodies against PML mutants. This shows that some peoplecarry antibodies specific for several residues in the sialic acidbinding site (e.g., patient 29 has antibodies to L55 and S269), whileothers have either antibody only to one site, L55 or 5269, and stillothers to no site. According to aspects of the invention, individualswho have no antibodies to the residues in the sialic acid binding siteor only to one of those residues might be more vulnerable to JCV escapemutants as they would have less protection from neutralizing antibodies.

TABLE 8 VLP EC50, patient ID dilution L55 S269 9 24820 + + 19 81850 − −22 14150 − + 29 6034 + + 31 802500 − − 33 870400 − − 39 − + 42 4161 − +49 5506 − − 51 51480 − − 59 2802 − − 60 2423 − −

Example 11 JC Virus (JCV) VP1 from Cerebrospinal Fluid (CSF) and Plasmaof Patients with Progressive Multifocal Leukoencephalopathy (PML) CarrySpecific Mutations of Amino Acid Residues Involved in Sialic AcidBinding

As described herein, PML is currently the second most frequent cause ofAIDS-related deaths. Unlike other opportunistic infections, it alsooccurs in HAART-treated patients, either shortly after starting orduring chronic successful treatment. Following primary infection, thecausative agent, JCV, establishes a persistent benign infection in theurinary tract and is excreted in urine in 30% of healthy persons. Themechanisms leading to JCV reactivation and PML are unclear, but it isknown that the major JCV capsid protein, VP1, is involved in cell entry,through binding with cell sialic acid residues and, recently, VP1 aminoacid substitutions have been reported in PML.

The entire JCV-VP1 region was amplified, cloned (2 to 48 clones persample, median 23) and sequenced from the CSF of 26 PML patients (20with HIV infection), and 11 paired plasma and 6 paired urine samples.From 9 patients, sequential CSF (n=7) or plasma (n=2) samples were alsoanalysed. JCV DNA was measured by real-time PCR. 3D modeling was used tomap the mutations on VP1 structure.

DNA Extraction and VP1 Amplification:

DNA was extracted from 200 μL of CSF, plasma or urine using the QIAampBlood Kit (Qiagen) and eluted in a final volume of 50 μL.

The entire JCV-VP1 region was amplified by nested PCR using thefollowing primers:

Outer (2027 bp) (SEQ ID NO: 55) VP1-LF GCAGCCAGCTATGGCTTTAC(SEQ ID NO: 56) VP1-LR GCTGCCATTCATGAGAGGAT Inner (1233 bp)(SEQ ID NO: 57) VP1-SF CCTCAATGGATGTTGCCTTT (SEQ ID NO: 58)VP1-SR AAAACCAAAGACCCCT

PCR reaction mixtures consisted of 5 μL of 10×PCR buffer, 4 mM of eachdNTP, 0.7 μM of primers VP1-LF and VP1-LR in the first round and primersVP1-SF and VP1-SR in the second round, 1.25 unit of Platinum Taq HF(Invitrogen) and 14 of extracted DNA in a total volume of 50 μL. Cyclingparameters were (for both first and second round) 30 cycles at 94° C.for 20 sec, at 58° C. for 30 sec and at 68° C. for 90 sec in anautomated thermal cycler (Applied Biosystems).

After the first amplification with the outer primers, 2.5 μl ofamplified product was transferred from the first to the second reactionmixture. Following amplification with the inner primers, 10 μl of theamplified product from the second mixture was electrophoresed on a 2%agarose gel containing 0.5 μg/ml ethidium bromide. The results werephotographed under U.V. illumination and regarded as positive when aband corresponding to the expected by long DNA fragment was present.

VP1 PCR Cloning:

The amplification product was purified by the Qiagen purification kit.A's were added to the ends of the cleaned up PCR product by Taqpolymerase (A-overhang reaction) and cloning was carried out by the TOPOTA cloning kit (Invitrogen). Mini-prep DNA was prepared (Qiagen) fromcolonies containing the cloned VP1 PCR product.

VP1 Sequencing:

Two to 48 clones were sequenced for each sample (median 23). Followingtranslation of the VP1 sequences, amino acid mutations were marked bycomparison to the large selection of VP1 sequences from PML and non-PMLcases. Only mutations present in more than one clone for sample wereconsidered.

Real-Time PCR for Quantification of JCV-DNA:

JCV DNA was quantified in CSF, plasma and urine samples by real-timePCR, as described previously (Bossolasco S, Calori G, Moretti F,Boschini A, Bertelli D, Mena M, Gerevini S, Bestetti A, Pedale R, SalaS, Sala S, Lazzarin A, Cinque P. Prognostic significance of JC virus DNAlevels in cerebrospinal fluid of patients with HIV-associatedprogressive multifocal leukoencephalopathy. Clin Infect Dis. 2005 Mar.1; 40(5):738-44.)

Patients:

PML patients were selected on the basis of the availability of either a)paired CSF and plasma or urine samples or b) sequential CSF samples, allwith detectable JCV DNA by real time PCR. Samples had been drawn frompatients followed at the Clinic of Infectious Dieaeses, San RaffaeleHospital, Milano, between 1993 and 2008. Sample aliquots were keptstored at −80° C. until the retrospective analyses for the presentstudy. JCV VP1 was successfully amplified from a total of 26 CSF, 11plasma and 6 urine samples from a total of 30 PML patients.

Analysis of Clinical Samples—Study Design:

1. Analyses of CSF sequences:

CSF sequences were examined from 26 patients (Table 9) and both type andfrequency of mutations, as well as their correlations with patientsvariables were analysed.

2. Analysis of sequences from paired samples from same patients:

Paired CSF/plasma/urine samples (“triplets”) were examined in 2patients. CSF/plasma, CSF/urine and plasma/urine pairs were examinedfrom, respectively, 6, 1 and 3 patients. Sequences obtained fromtriplets and pairs were compared.

3. Analysis of sequences from sequential samples:

Sequential CSF or plasma samples were available from 5 and 2 patients,respectively. These samples had been drawn close to the diagnosis of PML(baseline) and at different times afterwards. From each patient, 2 to 3samples drawn over a time frame of 17 to 477 days were analysed. 5 ofthese patients had a progressive course of PML. One patient experiencedvirological response after commencement of HAART. One patient underwentclinical and virological remission following treatment with cytarabine,but had a relapse of PML after 6 months and following withdrawal ofcytarabine.

TABLE 9 Characteristics of the 26 PML Patients with JCV VP1 analysis HIVstatus (pos:neg) 20:6 Median age 37 Sex (M:F) 18:8 JCV DNA copies/mL(median, IQR) 22,351 (6416-1,178,877) Ongoing HAART (number ofpatients) *  6 Progressors vs. survivors (number of patients) 18:2 *Refers only to patients with HIVrelated PML

Analysis of Clinical Samples—Results:

1. Analyses of CSF Sequences:

VP1 PML-specific mutations were defined as mutations that are notnormally present in the urine of patients without PML. These mutationsor deletions do not involve mutations at positions determining VP1genotypes—which distinguish VP1 sequences according to theirgeographical distribution.

One of 8 different PML-specific mutation or deletion was identified inCSF from 24 of 26 patients (92%). These involved amino acidsubstitutions in one of the JCV VP1 outer loops (Table 10).

In all of the cases almost all of the clones from the same samplecontained the mutation or the deletion. In 5 patients, two differentmutations or deletions were identified, either in same clones (n=2) orin different clones (n=3).

TABLE 10 JCV VP1 mutations in the CSF of patients with PML VP1 VP1mutation or Nr of Patients loop deletion (aa) patients HIV-pos (n = 20)BC 51-52 del 1  55F 5 55F + 271H* 1  61L 1 61L + 55 del* 1 DE 122R 2122R + 125-127 del 1 122R + 2V** 1 HI 265D 2 267F + 61L* 1 169F 2 0 2HIV-neg (n = 6) BC  55F 4 HI 265H 1 269F 1 The BC, DE and HI JCV VP1loops are defined by similarity of their aa sequences to the VP1 loopsof SV40 (Chang D, Liou Z M, Ou W C, Wang K Z, Wang M, Fung C Y, Tsai RT. Production of the antigen and the antibody of the JC virus majorcapsid protein VP1. J Virol Methods 1996 May; 59(1-2): 177-87), whichwere previously determined by X-ray crystallography (Liddington R C, YanY, Moulai J, Sahli R, Benjamin T L, Harrison S C. Structure of simianvirus 40 at 3.8-A resolution. Nature 1991 Nov 28; 354(6351): 278-84).*Either mutation/deletion was present in different clones (55F + 271H in15 and 4 clones; 61L + 55 del in 18 and 4 clones; 267F + 61L in 15 and 4clones) **Both mutations/deletions were present in each clone

Higher CSF JCV DNA levels were found in patients with mutations of theBC and HI loops than in those with mutations of the DE loop or nomutations (FIG. 19). No correlation was observed in patients withHIV-associated PML between type of mutation and CD4 cell count, plasmaHIV-1 RNA level or survival time.

2. Analysis of Sequences from Paired Samples:

The analysis of the 2 CSF/plasma/urine triplets showed the same VP1PML-specific mutation in CSF and plasma, whereas no PML mutation waspresent in the corresponding urine sequences (Table 11). Similarly,identical PML-specific mutations were found in CSF and plasma sequencesfrom 6 patients with CSF/plasma pairs (Table 12), but only in either CSFor plasma, but not in urine sequences of 1 patient with CSF/urine pairsor 3 patients with plasma/urine pairs (Table 13).

TABLE 11 JCV VP1 mutations in paired CSF/plasma/urine samples frompatients with PML Type of Pt Lab ID sample PML mutation 1 CSF 269F PLASMA* 269F URINE 0 2 CSF 269F PLASMA 269F URINE 0

TABLE 12 JCV VP1 mutations in paired CSF/plasma samples from patientswith PML Type of JCV DNA Pt Lab ID sample c/mL PML mutation 3 CSF 269FPLASMA 269F 4 CSF 269F PLASMA 269F 5 CSF 122R PLASMA 122R 6 CSF 0 PLASMA0 7 CSF  55F PLASMA  55F 8 CSF 269F PLASMA 269F

TABLE 13 JCV VP1 mutations in paired CSF/urine or plasma/urine samplesfrom patients with PML Type of Pt Lab ID sample PML mutation 9 CSF 55FURINE 0 10 PLASMA 122R  URINE 0 11  PLASMA** 55F URINE 0 12 PLASMA 0URINE 0

3. Analysis of Sequences from Sequential Samples:

Analysis of sequential CSF or plasma samples revealed the persistence ofthe PML-specific mutations in 7 patients with progressive disease andstable or increasing JCV DNA in CSF (Tables 14 and 15).

In the patient undergoing virological response, the principal PMLmutation present in the first CSF sample was no longer found in a secondCSF sample showing a decrease of JCV DNA level; in this latter sample,the emergence of a previously minor represented mutation was observed.

In the patient undergoing PML relapse a few months after clinical andvirological remission of a first episode of PML, two different mutationswere present in CSF samples drawn during the two episodes.

TABLE 14 PML Mutations in Patients with Sequential CSF Samples Daysafter Pt Lab ID first sample PML mutation A 0 55F 17 55F 92 55F B 0269F  208 265D  C 0 265D  56 265D  D 0 55F 66 55F E 0 122R + 2V ** 477122R + 2V ** F 0 51-52del 230 51-52del G 0 61L + 53-55 del* 63 53-55del

TABLE 15 PML Mutations in Patients with Sequential Plasma Samples Daysafter Pt Lab ID first sample PML mutation H 0 269F 21 269F I 0 267Y 56267YTables 14 and 15, notes:

* Either mutation/deletion was present in different clones (55F+271H in15 and 4 clones; 61L+55 del in 18 and 4 clones; 267F+61L in 15 and 4clones)

** Both mutations/deletions were present in each clone

Molecular Modeling of JCV-VP1:

Molecular Modeling of JCV VP1/Tetrasacharide Complex:

A homology model of the JCV VP1 protein pentameric unit was built withMODELER using the structure of MPyV VP1 (Protein Data Bank ID: 1VPS as atemplate. The model of NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NActetrasaccharide was build based on the structure ofNeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NAc bound to MPyV VP1. Themodel of the JCV VP1/NeuNAc-(α2,3)-Gal-(β1,3)-[(α2,6)-NeuNAc]-Glc-NActetrasaccharide was extensively refined in CHARMM and was analyzed usingPyMOL visualization software (The PyMOL Molecular Graphics System (2002)DeLano Scientific, Palo Alto, Calif., USA. http://www.pymol.org)

Accordingly, compared to wild-type virus (that present in urine ofhealthy persons) one of 8 specific single mutations or deletions wasidentified in almost all CSF clones from each of 24/26 patients (92%).These conferred substitutions or deletions in one of the three outerloops of VP1, most frequently involving residues 55 (55F, 7 patients,27%) and 269 (269F, 6 patients, 23%). Paired plasma always showed thesame CSF mutation, but no mutations were identified in urines. Mutationswere maintained in sequential samples from 7 patients with progressivedisease and stable or increasing JCV DNA in CSF. They were lost in 2patients: 1 undergoing PML remission and relapse, with onset of a newmutation; and 1 with decreasing CSF JCV DNA, with emergence of apreviously minor different mutant variant. By 3D modelling, all mutatedresidues clustered within or in the immediate proximity to the sialicacid cell receptor binding site on VP1.

Therefore, based on this data, in patients with PML, JCV found from CSFand plasma, but not urine, carries PML-specific VP1 substitutions. Thesesubstitutions are maintained during disease progression. They involvethe BC, DE or HI external loops of VP1, at critical sites for bindingwith the sialic acid cell receptor. Accordingly, in PML, JCV from CSFand plasma, but not urine, carries VP1 substitutions at critical sitesfor cell binding, which are maintained during the disease. Thesefindings support a model whereby JCV acquires adaptive changes duringtransition from sites of persistence to the brain, eventually leading toPML.

Example 12 Residues in the Sialic Acid Binding Pocket of VP1

Residues within 12 angstroms from the modeled sialic acid containingsugar (as described in the Examples herein) are listed in Table 16.

TABLE 16 MET 48 GLY 49 ASP 50 PRO 51 ASP 52 GLU 53 HIS 54 LEU 55 ARG 56GLY 57 MET 57 GLY 58 PHE 58 GLN 59 SER 59 LYS 60 PRO 60 SER 61 ILE 62PRO 63 SER 63 ILE 64 SER 65 SER 65 ASP 66 LEU 66 THR 67 THR 67 GLU 68PHE 68 GLU 69 GLY 69 GLY 70 SER 70 ASP 71 GLN 71 SER 72 TYR 72 PRO 73TYR 73 ASN 74 GLY 74 LYS 75 TRP 75 ASP 76 SER 76 ARG 77 MET 77 GLY 78LEU 78 ILE 79 PRO 79 ASN 80 LEU 81 ALA 82 THR 83 SER 84 ASP 85 THR 86GLU 87 ASP 88 SER 89 PRO 90 GLY 91 ASN 92 ASN 93 THR 94 LEU 95 PRO 96ASN 120 VAL 121 HIS 122 SER 123 ASN 124 GLY 125 ASP 130 ASN 131 GLY 132ALA 133 ALA 134 ASP 137 VAL 138 HIS 139 GLY 140 PHE 141 ASN 142 LYS 143THR 150 LYS 151 GLY 152 ILE 153 SER 154 PHE 159 ASN 160 TYR 161 ARG 162THR 163 THR 164 TYR 165 PRO 166 ASP 167 ASP 180 GLN 180 ARG 182 THR 183LYS 184 TYR 185 LYS 186 GLU 187 GLU 188 VAL 190 GLN 206 MET 262 PHE 263THR 264 ASN 265 ARG 266 SER 267 GLY 268 SER 269 GLN 270 GLN 271 TRP 272ARG 273 TRP 288 ARG 289 VAL 290 THR 291 ARG 292 ASN 293 TYR 294 ASP 295VAL 296 VAL 296 HIS 297 HIS 298 TRP 299 ARG 300

Example 13 CSF and Plasma, but not Urine JCV of PML Patients CarryPML-Associated VP-1 Mutations

To investigate whether PML-associated mutations are specificallyselected in CSF of PML patients, initially paired CSF and urinesequences from 7 patients were analysed (Table 17). In each patient, CSFJCV-VP1 carried one amino acid substitution that was not present in theurine derived sequence. CSF and urine sequences were otherwise identicalwithin individual patients and characterized by identical polymorphismsas compared to the reference strain (same virus subtype). PML-associatedCSF mutations involved codons 55, 60, 267, 269, and also codon 122.Plasma VP1 sequences, available from 13 patients (Table 17), were in allthe cases identical to the correspondent CSF-derived sequence, carryingthe same PML-associated substitution.

TABLE 17 Table 17. JCV VP1 amino acid substitutions in sequences derivedfrom paired urine, CSF and plasma samples from PML patientsPML-associated Matrix JCV subtype substitution 1 Urine 1B none CSF 1BL55F Plasma n.a. n.a. 2 Urine Mad-1 none CSF Mad-1 H122R; A2V Plasman.a. n.a. 4 Urine 1A none CSF 1A H122R Plasma 1A H122R 5 Urine Mad-1none CSF Mad-1 S269F Plasma Mad-1 S269F 6 Urine Undet. none CSF CONSS267Y Plasma Undet. S267Y 7 Urine 1B none CSF 1B S269F Plasma 1B S269F 8Urine 1A none CSF 1A S269F Plasma 1A S269F 9 Urine n.a. n.a. CSF Undet.S269F Plasma Undet. S269F 10 Urine n.a. n.a. CSF 2B S269F Plasma 2BS269F 11 Urine n.a. n.a. CSF 4v164K none Plasma 4v164K none 12 Urinen.a. n.a. CSF 1A L55F Plasma 1A L55F 13 Urine n.a. n.a. CSF 4v128A345KL55F Plasma 4v128A345K L55F 14 Urine n.a. n.a. CSF 1Bv117T S269F Plasma1Bv117T S269F 16 Urine 4v164K none CSF n.a. n.a. Plasma 4v164K L55F U,urine; CSF, cerebrospinal fluid; P, plasma. * when no substitution isidentified, then column value refers to nr of clones withoutsubstitution/nr of clones examined.

Example 14 CSF JCV of PML Patients Consistently Carry One of SeveralMutations or Deletions Located In Sites Critical for Cell Binding

To define type and frequency of VP1 PML-associated substitutions invivo, CSF-derived VP1 sequences in a larger group of patients wereanalysed. One main PML-associated mutation or deletion was identified in37 of 40 patients (90%) (Table 18). The VP1 gene was cloned andsequenced for a number of clones as described in Materials and Methods.Mutations were identified by comparing the sequences to either VP1sequences from matched urine samples (when available) and to 460sequences isolated from the urine of non-PML individuals (n=460) asreported in the Genebank. In some patients the mutation H122R wasidentified. In some patients the mutation 2831 was identified. In somepatient the mutations A2V was identified. In some patient the deletion50-51 was identified. In some patient the deletion 50-51 was identified.In some patient the deletion 54-55 was identified. In some patient thedeletion 123-125 was identified. In some patient the deletion 125-134was identified. In some patient the deletion 126-134 was identified.

The most frequent changes involved codons 55 and 269, each identified in25% of the patients. In addition to the substitutions already reported,at codons 55, 60, 61 and 265, 267, 269, several other PML-associatedmutations or deletions were newly identified, involving non polymorphicVP1 codons as evidenced by comparison to urine derived sequences fromthe Genebank. All these newly identified mutations are also located incritical VP1 binding sites (Table 18). FIG. 20 is a structural model ofJCV VP1/NeuNAc-(a2,3)-Gal-(b1,3)-[(a2,6)-NeuNAc]-Glc-NAc tetrasaccharidecomplex. (A). Surfaces of five chains of JCV VP1 are shown. The RG motifessential for binding of core sialic acid is shown. PML-associatedmutated residues also are shown (L55, K60, S265, S269). Additionalmutations unique to PML-isolated samples also are indicated (S61, D66,Q271) and (P51, D52, H122, S123, N124, G125, Q126, A127). (B). Aclose-up view of FIG. 20A. The location of V296 of MPyV VP1 which ispredicted to be equivalent to S269 of JCV VP1 is shown in mesh.

TABLE 18 JCV VP1 PML-associated aa substitutions and deletions in CSF ofPML patients PML-associated substitution JCV VP1 subtype or deletion 14v-164K none 2 1Av-128S none 4 2B 50-51 del D66G N124S 5 1B L55F 6 1AL55F 7 3 L55F Q271H 8 1B L55F 9 1A L55F 10 4v-128A345K L55F 16 1B S61L54-55 del 18 1A H122R 19 1A H122R A2V 17 1B 123-125 del 20 4v-128A N265D21 4v-128A N265D 23 1B S267F S61L Q271H 24 2B S267F 25 Undet. S269F 271B S269F 31 2B L55F 34 2B N265H 36 2B S269F 37 1Bv117T S269F 38 1B S269F39 1A S269F Undet., undetermined; Cons., consensus; v, variant a.sequence available up to position 295

Example 15 Relationship between JC Virus Isolated from DifferentCompartments

To inquire into the relationship between viral populations in threecompartments (kidney, plasma and CSF) population variation of VP1sequences represented by individually sequenced clones from all threecompartments in three patients were analyzed. With the exception ofamino acid changes presumably associated with PML development, dominantVP1 genotypes in all three compartments were always identical. Allobserved sequence variation was due to low frequency single nucleotidevariants. This suggests that PML causing virus originated frompreexisting resident population in kidney.

The relationship between viral populations in the three compartments canbe characterized by the degree of shared low frequency polymorphism.Single nucleotide variants observed in two or more clones were analyzed.In all three patients, the majority of genetic variation was confined toindividual compartments as allelic variants present in more than onesequence were observed in a single compartment. Therefore, the viralpopulation is highly structured.

In two of the three patients several allelic variants were sharedbetween CSF and plasma populations and no variant was shared between CSFand kidney populations or plasma and kidney populations. In each ofthese two patients, the same PML-associated mutation was observed inclones from CSF and plasma. These data are consistent with a singleorigin of the CSF and plasma populations, presumably due to a one-timeescape from kidney event. In this scenario sequence variation sharedbetween the CSF and plasma populations originated after the fixation ofthe PML-associated mutation outside of kidney (assuming that independentmultiple mutations in the same site are not likely). VP1 sequencevariation shared between CSF and plasma populations may indicatepresence of viral migration between compartments. It is also consistentwith a scenario of step-wise infection, where a resident viralpopulation outside of kidney first establishes in one compartment(either CSF or plasma), accumulates genetic variation, and then infectsthe second compartment.

A third patient presents a different picture of shared polymorphismbetween compartments. Two variants were shared between CSF and kidney,three variants were shared between plasma and kidney and one variant wasshared between all three compartments. All clones isolated from CSF andmost of clones isolated from plasma harbored the same PML-associatedmutation (S269F). However, three clones isolated from plasma had adifferent PML-associated mutation (S267F). These clones lacked thedominant PML-associated mutation.

Although the possibility of multiple successive infections of CSF andplasma of Patient 3 cannot be excluded, these observations are notnecessarily inconsistent with the scenario of a single origin of CSF andkidney populations after a one-time escape from kidney. The data forPatient 3 might be an example of a soft selective sweep. Soft selectivesweep unlike the hard selective sweep does not eliminate preexistinggenetic variation completely. The soft sweep scenario is very likely ifthe product of effective population size and mutation rate (Ne×mu)exceeds one. In this case multiple instances of a beneficial mutation(or several beneficial mutations of equal or comparable selectiveadvantage) likely existed in the original population on differenthaplotypic backgrounds. After a soft sweep, all sequences would harbor abeneficial mutation but the population remains polymorphic. It ispossible that multiple instances of S269F mutation and S267F mutationexisted in the kidney population before the escape and the fixationoccurred following the soft sweep scenario leading to the co-existenceof S269F and S267F VP1 variants in the plasma population and sharedvariation between all three compartments.

Example 16 Correlation of Various Mutations with the Clinical Outcome

To assess whether individual PML-associated VP1 substitutions could beclinically significant, e.g., being differently selected according topatient and clinical context or associated with different diseaseoutcome, their presence was correlated to a number of variables.

It was observed that JC virus with no VP1 substitutions or substitutionsinvolving positions 122-134 was present at the significantly lower DNAlevel in CSF as compared to virus with substitutions involving otherpositions (p=0.013, Mann-Whitney test), e.g., with substitutions eitherat position 50-61 (p=0.02) or 265-269 (p=0.036) s carrying different VP1substitutions.

Example 17 PML Associated Mutation Decrease Ability of VP1 toHemagglutinate RBCs

In order to understand the role PML associated mutations play in diseasepathogenesis, the effect of some of these mutations on viral receptorbinding was investigated. Since JCV is known to bind to sialic acidstructures (Liu), viral ability to cause hemagglutination of RBCs thatexpress those structures has been widely used as a model for viralinteraction with its receptor.

Viral like particles (VLPs) prepared from the major JCV capsid proteinVP1 has been widely used as a good model investigating the interactionsof polyomaviruses with their cell receptors. Similar to viral capsid,VLPs form viral capsid structure as evidenced from electron microscopy(data not shown). Next, binding of VLPs prepared from either a “wildtype” or various “mutant” VP1 molecules was investigated. A number ofPML-associated mutations that were shown to be positively selectedduring PML were introduced as point mutations on a backbone of a VP1molecule from a single viral background (JCV type 3) and compared theirability to hemagglutinate (e.g., bind) red blood cells (RBC). VLPsprepared from VP1 molecule of type 3 virus just like for VLPs preparedfrom type 1A (e.g., Mad-1) virus can cause RBC hemagglutination, andthey do that at the concentration as low as 760 pg/ml. However, VLPscarrying one of most frequently occurring mutations 55F, 267F andmutations at position 269 (F or Y) did not cause hemagglutination evenat the highest tested concentration of 100 μg/ml, which corresponds tomore than 100,000-fold decrease in activity. Hemoagglutination was stillapparent with the 60E, 265D and 271H mutant VLPs, but only at very highconcentrations that corresponded to 200 to 25,000-fold losses inactivity. Only binding of 66H mutant VLP was not strongly affected andshowed only 3-fold decrease in hemoagglutination (3000 pg/ml as thelowest hemagglutinating concentration). Neither 66H nor 271H mutationswere observed among the sequences analysed in this example, but thesemutations are identified to be PML-associated, based on an analysis ofsequences from Genebank. This effect of mutations on the ability of VLPsto cause hemagglutination suggests that the mutations may change viralreceptor specificity by abrogating the ability of virus to bind to cellreceptor(s).

Example 18 PML-Associated Mutations Lose Ability to Hemagglutinae RBCsfrom Different Blood Groups

VLPs carrying PML-associated mutations lose ability to hemagglutinaeRBCs from different blood groups. For hemagglutination assay red bloodcells (RBCs) were incubated with serial two-fold dilutions of variousVLPs starting from 100 μg/ml. Minimum HA concentration is the lowestconcentration of VLP protein that still agglutinated RBCs.Hemaglutination was examined by visual inspection as previouslydescribed. All hemagglutination reactions were conducted in duplicates.Mean+/−SD for the minimum HA concentration is calculated based on thehemaglutionation results from four different blood group donors A, B, Oand AB). VLPs displaying lowest hemagglutination concentration of 100μg/ml did not cause any hemagglutination at highest VLP concentrationtested (e.g., 100 μg/ml).

Example 19 PML Associated Mutations Change Ganglioside Specificity ofVP1

In order to further dissect JCV VP1 receptor specificity binding ofvarious mutant VLPs to different gangliosides was investigated.Gangliosides are a group of complex glycosphingolipids in whicholigosaccharide chains containing one or more sialic acids(N-acetylneuraminic acid, NeuNAc) are attached to a ceramide, whichanchors the structure to cellular membrane. VLP binding to theseganglioside was measured in ELISA like format, where gangliosides werecoated in ELISA plates akin to antigen and followed by addition of VLPs,which binding was detected with VP1 specific monoclonal antibodies.Using a type 3 “wild type” JCV VLP, the strongest binding was observedto GD1b, GD2 and GT1a, with more than 8-12 fold increase of signal ascompared to the background produced by binding of VLP to the wellwithout any ganglioside (FIG. 21). PML specific mutations of VP1 abolishor drastically change specificity of viral capsid protein VP1 forsialated gangliosides. Binding of VLPs to an array of gangliosidescoated on a 96-well plate was detected with a two step process involvingdetection of VLPs bound to a ganglioside with VP1-specific murineantibodies and anti-murine IgG HRP labeled antibodies followed bydevelopment with TMB solution. VLP binding to a specific ganglioside wascalculated as percent increase in the optical density obtained with thespecific VLP present relative to that obtained without VLP andantibodies alone in the presence of the same galnlioside,100*(OD₄₅₀(plus VLP)−OD₄₅₀(minus VLP))/OD₄₅₀(minus VLP). Schematicstructure of ganglioside is shown to reveal core binding structure boundby various VLPs. One representative experiment of four conducted isshown. This ganglioside specificity is consistent with what waspreviously described for the type 1A “wild type” virus Mad-1. Binding ofVLPs to asialo-GM1 and GD1a was much weaker but still significant overthe background, while binding to other gangliosides was very negligiblerelative to no-ganglioside background. Based on the binding pattern itappears that the major “core” structure bound by wild type virusconsists of a tetrasaccharide GalNAc(β1-4)[Neu5Ac(α2-8)-Neu5Ac(α2-3)]Gal(β1-4)Glc structure, with di-sialic acid motif ofNeu5Ac(β2-8)-Neu5Ac(α2-3) being crucial for binding.

However, binding of all mutant VLPs was drastically different from the“wild type” molecule. Specifically, as seen in FIG. 21, the VLP mutants55F, 60E, 267F, 269F and 269Y completely lost binding to all sialatedgangliosides while still showing unchanged binding to asialated GM1structure (e.g., asialo-GM1). Still, others, such as 66H, 265H and 271Hshowed a much broader range of ganglioside binding than the originalnon-mutated VLP molecule.

Example 20 Binding of VLPs to Various Cellular Targets

Results from hemagglutination and direct ganglioside binding assaysjointly suggest that PML associated mutations change viral receptorspecificity and especially viral ability to bind to sialatedoligosaccharide structures. Therefore, it was investigated next howthese mutation-conferred changes in specific receptor binding affectedJCV ability to bind to its purported target cells. Binding of the abovewild-type and mutant VLPs to the three major cell types reported to beimportant for viral cell cycle: kidney epithelial cells, lymphocytes andCNS cells was measured. Primary human renal proximal tubular epithelialcells (HRPTECs) were chosen as cells from the site of viral asymptomaticpersistence of non-mutated virus. Primary human B lymphocytes were alsochosen, as the cell type suggested to be critical for viral spread fromperipheral site to the CNS, as well as other lymphoid cells, e.g.,primary T lymphocytes and lymphocytic cell line Jurkat. Althoughproductive infection of lymphoid cells has not been unequivocallydemonstrated, lymphocytes have been shown to bind JCV via their sialicacid structures (Atwood et al.) and thus could potentially carry thevirus in vivo. Primary human fetal astrocytes and the human glial cellline SVG-A were also employed as a model cell type for CNS infectionduring PML. Infection of astrocytes has been well documented in PMLpatients and both of these glial cell types can be infected by JCV invitro.

PML-ogenic mutants of VP1 lose binding ability to kidney and blood cellsbut bind to the glial cells. Glial cell line SVG-A(A), primary humanastrocytes (B), human brain microvascular endothelial cell (C), kidneytubular epithelial cells (D), or peripheral blood mononuclear cells (Eand F) cells were first incubated with different VLP (as indicated onthe x-axis) and further incubated with anti-VP1 antibodies followed bystaining with fluorescently labeled antibodies. Binding of VLPs to T-(E) and B-lymphocytes(E) was evaluated after co-staining of PBMCs withantibodies specific for human CD3 and CD20 markers and gating on thecorresponding population. Ratio of mean fluorescent intensity (MFI) ofcells stained with anti-VP1 antibodies in the presence of VLPs relativeto the background MFI of cells stained only with the detectionantibodies in the absence of VLP is plotted for each VLP. Mean MFIratio+/SD is calculated based on the results from several independentexperiments (N is indicated on the graph). VLPs made from “wild type”VP1 strongly bound to all of the above cell types with MFI of more than2-fold above background staining. However, binding of mutant VLPs wasdependent on both the cell type and the position of the mutated aminoacid. Specifically, the most frequent PML associated mutations 55F,267F, 269F and 269Y abrogated binding of VLPs to kidney tubularepithelial cell and all lymphocytic cells, but not to the primaryastrocytes or glial cell line SVG-A. Both CNS cell types still stronglybound VLPs carrying these mutations (with the exception of 55F that bindSVG-A cells but not primary astrocytes). VLPs carrying 60E, 66H and 271Hstill bound all cell types, including kidney epithelial cells andlymphocytes, although binding of 271H was strongly diminished.

Example 21 VLPs Carrying PML-Associated Mutations Bind Glial Cells inSialic Acid Independent Fashion

Since binding of the virus to cells has been previously demonstrated tobe sialic acid dependent (e.g., Atwood et al), but the data demonstratethat most PML-associated mutations abolish binding of VLPs to sialicacid containing gangliosides, it was tested next whether binding ofmutant VLPs to cells is still sialic acid dependent. To do that eitherSVG-A or Jurkat cells were treated with Neuraminidase to remove sialicacid from cell surface. Staining of the cells with lectins specific forvarious conformations of neuraminic acids (e.g., Sambucus Nigra Lectin(SNA) and Maackia amurensis lectin (MAA)) proved neuraminidase treatmenteffectiveness in sialic acid removal. Binding of PML-ogenic mutants ofVP1 to glial cells is sialic acid independent. Glial cell line SVG-Acells or lymphoid cell line Jurkats were first either pretreated withα2-3,6 neuraminidase for 60 min at 37° C. or mock treated followed byincubation at 4C with the indicated VLP and staining with the detectionantibodies as described herein.

Binding of type 3 wild type VLPs to both SVG-A and Jurkat cells issialic acid dependent, confirming what was previously shown for Mad-1virus (e.g., type 1A wild type). However, binding of all tested but K60Emutant VLPs to SVG-A cells was not affected by neuraminidase treatmentsuggesting that binding of these mutants to these cells is sialicacid-independent. Interestingly, those mutants that were still able tobind to Jurkat cells (e.g., K60E, D66H, N265D and Q271H) bound in asialic acid dependent manner as evidenced by diminished binding toJurkat cells treated with the neuraminidase.

As can be seen from summary Table 19, binding of VLPs to Jurkat cellsappears to be largely sialic acid mediated, so that mutant VLPs that donot show any binding to sialated gangliosides do not bind to Jurkatcells and those that do also bind to Jurkat cells in sialic aciddependent fashion. One notable exception to that observation is N265Dmutant, which, based on direct binding to gangliosides, seemed to havegained very broad sialic acid binding, but still lost its binding toJurkat cell. Based on the above results it appears that VLP bindingresults are in line with VLP binding to RBCs as judged fromhemagglutination results.

In summary, it appears that PML associated mutations largely abolishsialic acid dependent binding of the virus to many different peripheralcells types, including RBCs, kidney tubular epithelial cells andlymphoid cells. Binding of mutant virus to CNS-specific glial cellsappears to be largely unaffected and sialic acid independent.

TABLE 19 Correlation of VLP binding between various assays SA-dependentHA SA HRPTEC Jurkat Astrocytes SVG-A Jurkat SVG-A Wild Type 3 +++ ++ ++++++ +++ +++ YES YES L55F − − − − +/− +++ NO NO K60E − − +++ ++ ++++ +++YES Part. D66H ++ +++ +++ +++ +++++ +++ Part. NO N265D + ++++ − − +++++++ NO NO S267F − − − − ++ ++ NO NO S269F − − − − ++ +++ NO NO S269Y −− − − ++ +++++ NO NO Q271H +/− +++ + + +++ +++++ YES NOThe following methods and materials were used in connection withexamples 13-21:

Patients and Samples

The present study was approved by the Ethical Committee of theInstitution. To investigate the presence and evolution of PML-associatedmutations in vivo, from a large cohort of PML patients, followed at theDepartment of Infectious Diseases of San Raffaele Hospital between 1992and 2009, 40 patients were initially selected from whom paired CSF,plasma or urine samples were available and contained JCV DNA asdetermined by real-time PCR (Bossolasco). VP1 was eventually amplifiedfrom at least two different types of samples in 14 of these patients.

43 additional PML patients were subsequently selected with only CSFavailable and JCV DNA detected in CSF by real-time PCR. JCV VP1 wassuccessfully amplified from 28 of these patients. Thus, paired samplesfrom 14 patients and CSF-derived sequences from a total of 40 patientscould be studied.

JCV VP1 PCR DNA Extraction and VP1 Amplification

DNA was extracted from 200 μL of CSF, plasma or urine using the QIAampBlood Kit (Qiagen) and eluted in a final volume of 50 μL. VP1 wasamplified either using primers flanking the whole VP1 gene (full VP1PCR), or, when amplification with this method was not successful, by asemi-nested PCR assay that amplified separately shorter VP1 regions(short fragment VP1 PCR).

The full VP1 PCR consisted of a nested assay that used the outer primersVP1-LF and VP1-LR, amplifying a 2027 bp fragment; and the inner primersVP1-SF and VP1-SR, amplifying a 1233 bp long fragment. The PCR reactionmixture consisted of 5 μL of 10×PCR buffer, 4 mM of each dNTP, 0.7 μM ofprimers VP1-LF and VP1-LR in the first round and primers VP1-SF andVP1-SR in the second round, 1.25 unit of Platinum Taq HF (Invitrogen)and 1 μL of extracted DNA (first round) or 2.5 μl of amplified product(second round) in a total volume of 50 μL. Cycling parameters were (forboth first and second round) 30 cycles at 94° C. for 20 sec, at 58° C.for 30 sec and at 68° C. for 90 sec in an automated thermal cycler(Applied Biosystems).

The short fragment PCR consisted of a semi-nested PCR that used primersVP1-1 and VP1-4a in the first round, amplifying a 797 bp fragment,followed by two semi-nested assays with primers VP1-1 and VP1-2a,amplifying a 481 bp-long fragment, or with primers VP1-1.5 and VP1-4a,amplifying a 490 bp-long fragment (Zheng) (Table 20) The PCR reactionmixture consisted of 2.5 μL 10×PCR buffer, 200 μM of each dNTP, 1.5 mMMgCl2, 0.5 μM primers and 1.25 unit of AmpliTaq Gold DNA Polymerase(Applied Biosystems). In the first round 4 μL of extracted DNA wereadded in a total volume of 25 μL and the cycling parameters were 20cycles at 94° C. for 20 sec, at 55° C. for 30 sec and at 68° C. for 90sec in an automated thermal cycler (Applied Biosystems). This first stepPCR product was purified with ExoSAP-IT PCR Clean-up Kit, the protocolconsists of a single pipetting step (enzyme mixture addition), a 30-minincubation at 37° C. followed by enzyme inactivation at 80° C. for afurther 15 min. In the second round of semi-nested PCR 4 μL of cleanedPCR product were added in a total volume of 25 μL and the cyclingparameters were 40 cycles at 94° C. for 20 sec, at 62° C. for 30 sec andat 68° C. for 90 sec in an automated thermal cycler (AppliedBiosystems).

With both assays, following amplification with the inner primers, 10 μlof the amplified product from the second mixture was electrophoresed ona 2% agarose gel containing 0.5 μg/ml ethidium bromide. The results werephotographed under U.V. illumination and regarded as positive when aband corresponding to the expected by long DNA fragment was present.

TABLE 20 Nucleotide sequences of the primers used in the full lengthnested PCR and short-fragment semi-nested PCR forJCV-VP1 Name SequenceNt. Number * VP1-LF GCAGCCAGCTATGGCTTTAC (SEQ ID NO: 55) VP1-LRGCTGCCATTCATGAGAGGAT (SEQ ID NO: 56) VP1-SF CCTCAATGGATGTTGCCTTT(SEQ ID NO: 57) VP1-SR AAAACCAAAGACCCCT (SEQ ID NO: 58) VP1-1TTGACTCAATTACAGAGGTAGAAT (SEQ ID NO: 59) VP1-4a AGAAATTGGGTAGGGGTTTTTAAC(SEQ ID NO: 60) VP1-2a AGGTACGCCTTGTGCTCTGTGTTC (SEQ ID NO: 61) VP1-1.5GTGCAGGGCACCAGCTTTCATT (SEQ ID NO: 62) * according to the Mad-1 strain(ref)

VP1 PCR Cloning and Sequencing

The amplification product was purified by the Qiagen purification kit.A's were added to the ends of the cleaned up PCR product by Taqpolymerase (A-overhang reaction) and cloning was carried out by the TOPOTA cloning kit (Invitrogen). Mini-prep DNA was prepared (Qiagen) fromcolonies containing the cloned VP1 PCR product. Two to 48 clones weresequenced for each sample (median 23). Following translation of the VP1sequences, amino acid mutations were marked by comparison to the largeselection of VP1 sequences from PML and non-PML cases. Only mutationspresent in more than one clone for sample were considered.

Real-Time PCR for Quantification of JCV-DNA

JCV DNA was quantified in CSF, plasma and urine samples by real-timePCR, as described previously (Bossolasco S. et al. 2005)

Hemagglutination Assay

Red blood cells (RBCs) from type O+ donors were washed twice andsuspended in Alsever's buffer (20 mM sodium citrate, 72 mM NaCl, 100 mMglucose, pH 6.5 adjusted with acetic acid) at a final concentration of˜0.5%. Serial two-fold dilutions starting from 100 μg/ml of VLPs wereprepared in Alsever's buffer and an equal volume of RBCs was added intoeach well of a 96-well “U” bottom microtiter plate and incubated at 4°C. for 3-6 hr (24 two-fold dilutions were performed). Minimum HAconcentration is the lowest concentration of VLP protein that stillagglutinated RBCs.

Ganglioside ELISA

Specific gangliosides (resuspended in methanol) were coated onto amicrotiter plate (10 μg) overnight. The plates were blocked (1% BSA(Fraction V), 0.1% Tween-20, PBS with Ca++, Mg++). VLPs were prepared at30 μg/ml in block buffer and added (100 μl/well). All incubations wereperformed on ice. After 90 minutes, the plates were washed with blockbuffer. VLP binding was detected with a two step process involvingbinding of anti-VP1 (PAB597 at 2 μg/ml) and HRP-anti-mIgG (1:100).Plates were washed and developed with TMB Turbo ELISA solution. Thereaction was stopped with acid and the plate read on a spectrophotometerat 450 nm.

Flow Cytometry Analysis of VLP Binding to Cells

Cells were detached and collected (SVG-A, Jurkat, Human Astrocytes,Human Renal Proximal Tubular Epithelials). A sample (1-5×10⁵ cells) wasfirst incubated with VLP (10-30 μg/ml) in FACS Buffer (1% BSA [FractionV], 2 mM sodium azide, PBS with Ca++, Mg++) in a volume of 50 μl for60-90 minutes on ice. Cells were washed with FACS Buffer and furtherincubated with anti-VP1 (PAB597 2 μg/ml) for 60 minutes, followed bywashing and incubation with AlexaFluor488-anti-mIgG (1:100) for another30-45 minutes. Cells were washed and fixed in Cytofix/Cytoperm for 20minutes, followed by a final wash and resuspension in FACS buffer. Thecells were analyzed on a BD FACSCalibur. Appropriate controls andstaining with other antibodies (isotype control, GM1, asialo GM1, GD1a,GT1b) and lectins (Peanut Agglutinin (PNA), Sambucus Nigra Lectin (SNA),Maackia Amurensis Lectin II (Mal II)) where necessary. In some casescells were pretreated with α2-3 neuraminidase, α2-3,6 neuraminidase, orPNGaseF to alter their cell surface suger structures.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety.

1. A method comprising: interrogating a biological sample from a subjectfor at least one indicium of a variant JCV VP1 capsid protein thatcomprises a substitution of amino acid residue 122, and wherein thesubject is determined to have an increased susceptibility for PML if thesample comprises the at least one indicium.
 2. The method of claim 1,further comprising interrogating the biological sample for at least oneindicium of a variant JCV VP1 capsid protein that comprises asubstitution of at least one of amino acid residues 2, and
 66. 3. Themethod of claim 2, wherein the sample is interrogated using an assaycapable of detecting at least one indicium of each of variant JCV VP1capsid proteins having a substitution of at least one of amino acidresidues 2, 66, and 122, and wherein the subject is determined to havean increased susceptibility for PML if the sample comprises at least oneindicium of at least one of the variant JCV VP1 capsid proteins.
 4. Themethod of claim 1, further comprising interrogating the biologicalsample for at least one indicium of a variant JCV VP1 capsid proteinthat comprises a deletion of one or more of amino acid fragments 50-51,54-55 and 123-125.
 5. The method of claim 4, wherein the sample isinterrogated using an assay capable of detecting at least one indiciumof each of variant JCV VP1 capsid proteins having a deletion of at leastone of amino acid fragments 50-51, 54-55 and 123-125, and wherein thesubject is determined to have an increased susceptibility for PML if thesample comprises at least one indicium of at least one of the variantJCV VP1 capsid proteins.
 6. The method of claim 1, further comprisinginterrogating the biological sample for at least one indicium of avariant JCV VP1 capsid protein suspected of having low sialic acidbinding.
 7. The method of claim 1, further comprising interrogating thebiological sample for at least one indicium of a variant JCV VP1 capsidprotein that comprises a substitution of at least one of amino acidresidues 55, 60, 265, 267, and
 269. 8. The method of claim 7, whereinthe sample is interrogated using an assay capable of detecting at leastone indicium of each of variant JCV VP1 capsid proteins having asubstitution of at least one of amino acid residues 55, 60, 265, 267,and 269, and wherein the subject is determined to have an increasedsusceptibility for PML if the sample comprises at least one indicium ofat least one of the variant JCV VP1 capsid proteins.
 9. The method ofclaim 1, wherein the biological sample is a blood sample.
 10. The methodof claim 1, wherein the biological sample is a CSF sample.
 11. Themethod of claim 1, wherein the biological sample is a urine sample. 12.The method of claim 1, wherein the subject is known to have beenpreviously infected with a wild-type JCV.
 13. The method of claim 12,wherein a new biological sample from the subject is interrogated for atleast one indicium of at least one variant JCV VP1 capsid protein atleast twice each year.
 14. The method of claim 1, wherein the detectionof at least one indicium of a variant JCV VP1 capsid protein is used toidentify the subject as inappropriate for an immunosuppressivetreatment.
 15. The method of claim 1, wherein the detection of at leastone indicium of a variant JCV VP1 capsid protein is used to recommend amodification of an immunosuppressive treatment for the subject.
 16. Themethod of claim 1, wherein the absence of indicia of a variant JCV VP1capsid protein is used to identify the subject as appropriate for animmuno suppressive treatment.
 17. The method of claim 1, wherein theabsence of indicia of a variant JCV VP1 capsid protein is used toidentify the subject as appropriate for continued immunosuppressivetreatment.
 18. The method of claim 1, wherein the biological sample isinterrogated for the presence of an antibody that is specific for avariant JCV VP1 capsid protein.
 19. (canceled)
 20. The method of claim1, wherein the biological sample is interrogated for the presence of avariant JCV VP1 capsid protein.
 21. The method of claim 1, wherein thebiological sample is interrogated for the presence of a nucleic acidsequence that encodes a variant JCV VP1 capsid protein.
 22. The methodof claim 20, wherein the biological sample is interrogated using anELISA based analysis.